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Solid lipid nanoparticles (SLN) are nanocarriers in the 10–1000 nm range of a solid core, containing both hydrophilic and hydrophobic active pharmaceutical ingredients. SLNs are composed of well-tolerated and biodegradable solid lipids such as mono-, di-, and triglycerides, fatty acids, waxes, and steroids, as well as lipophilic and hydrophilic emulsifying agents. This composition of biocompatible molecules makes SLNs one of the most successful options for the administration of drugs with different routes of administration. To determine its size, morphology, and surface charge, laser diffraction spectroscopy techniques, dynamic light scattering, coulter counter, scanning ion occlusion sensing, and advanced microscopy techniques such as scanning electron microscopy, transmission electron microscopy, and atomic force microscopy are some of the most widely used methods. Surface morphology and length can be measured by electron microscopy, while dynamic light scattering and photon correlation spectroscopy determine particle size and size distribution. In addition, colloidal stability can be determined by zeta potential analysis, indirect measurement of surface charge, and differential scanning calorimetry to characterize particles and drug interactions.
Faculty of Chemistry Sciences, Autonomous University of San Luis Potosi, Mexico
Gabriela Navarro-Tovar
Faculty of Chemistry Sciences, Autonomous University of San Luis Potosi, Mexico
Elvia Zárate-Hernández
Faculty of Chemistry Sciences, Autonomous University of San Luis Potosi, Mexico
Patricia Aguirre-Bañuelos*
Faculty of Chemistry Sciences, Autonomous University of San Luis Potosi, Mexico
*Address all correspondence to: paguirreb@uaslp.mx
1. Introduction
Pharmaceutical nanocarriers comprise nanoparticles, nanospheres, nanocapsules, nanoemulsions, nanoliposomes, and nanoniosomes [1]. Solid lipid nanoparticles (SLN) can be defined as solid lipid colloidal particles composed of organic matter and carbon in a range of 10–1000 nm in which the active pharmaceutical ingredients (API) are dissolved or encapsulated in lipids (Figure 1) [2, 3, 4]. The underlying mechanism for the formation of lipid nanocarriers is hydrophilic-hydrophobic interactions and van der Waals forces between phospholipids and water molecules [5]. In addition, the physicochemical properties of lipids, such as biocompatibility, low susceptibility to erosion phenomena, and slow water absorption, make lipids an ideal nanocarrier system to improve aqueous solubility and bioavailability of APIs [6, 7].
Figure 1.
General diagram of the solid lipid nanoparticles.
Some advantages of SLNs as a vehicle for APIs include: covering the bitter taste of the drug in oral administration; maintenance of therapeutic drug concentrations and circulatory time at target sites; protection against premature degradation in the gastrointestinal tract; improved pharmacokinetics, solubility, bioavailability, and stability; reduced toxicity; dose reduction and dose frequency; improvement of patient compliance; and prevention, reduction or delay of the onset of resistance, in addition to the fact that the by-products or metabolites of SLNs are significantly safe and can be easily eliminated through the normal process of excretion [8].
SLNs have been sought as a means to improve the solubility and bioavailability of many drugs, both hydrophilic and lipophilic, especially drugs belonging to class two (high permeability and low solubility drugs) and four (low permeability and low solubility drugs) of the Biopharmaceutical Classification System (BCS) [9].
SLNs have been developed for various applications, including nutraceuticals, cosmetics, pharmaceuticals, and biomedicals, as they can transport a variety of components, including small drug molecules, large biomacromolecules (polysaccharides, etc.), genetic material (DNA/RNA) [10], vaccine antigens [11], antineoplastic [12, 13], antimicrobial [14], they can also be applied in for the targeted delivery of brain medications since enhancing the ability of the drug to penetrate through the blood-brain barrier (BBB) [15].
By focusing on cellular delivery, SLNs can enhance drug delivery to target cells by various mechanisms, such as passive mechanisms that take advantage of the tumor microenvironment, active mechanisms by surface modification of the SLN, and the co-distribution mechanism. SLNs can combine many different drugs and be effective in various types of tumors (i.e., breast, lung, colon, liver, and brain), supporting their potential [16].
SLNs provide several indirect ways to address resistance problems, such as achieving a sustained release profile of a drug, maintaining concentrations within its therapeutic range, and thus avoiding potential adverse effects. Suboptimal levels can promote the selection of resistant bacteria, reduce drug toxicity by encapsulation, permitting higher doses. Promote accumulation in target cells using active targeting, and increase the inhibitory effect (i.e., decrease MIC) on bacterial strains [17].
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, 28, 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.
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 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.
As mentioned above, SLNs are composed of a lipid substrate surrounded by stabilizers and one or more active molecules incorporated into these particles. Thus, the structure of a particle consists of an outer layer that determines its surface properties and an inner layer or core material that determines factors such as the size, shape, and location of the active ingredient [32]. Therefore, how the lipid substrates stabilizers and active components that make up the colloidal lipid system are organized and interconnected determines their behavior as drug transporters and is strongly influenced, among other factors, and the crystalline behavior of lipids used.
It has been reported that under heat treatment or during storage, polymorphic transformations can occur, and the particles can change from spherical shapes to platelets. Platelet forms are associated with the stable polymorphic state β for the solid lipid of the matrix, and spheroidal or disc-shaped records are associated with the polymorphic state α [33].
In general, two structure models have been proposed for SLN, in which it is assumed that the particles are surrounded by the surfactant that forms the outer layer. The first is that the molten lipid droplets solidify during cooling, maintaining their spherical shape. On the other hand, the second model proposes that the cooling of molten lipids produces a flat laminar structure with surfaces structured by folds, edges, and steps that occur during the recrystallization process and where the lipid structure from polymorph α to polymorph β comes from stable [28].
3.1 API localization within SLNs
The location of the active molecule depends on the structural organization of the colloidal lipid systems; in this way, from the model corresponding to the basic spherical model for SLN, three general and commonly used classifications emerge: (i) active molecule is homogeneously distributed along the length of the particle structure (homogeneous matrix); (ii) the API is concentrated within the particle and is surrounded by the lipid matrix in a core-shell structure (enriched core); and (iii) the API is concentrated on the surface of the particle (enriched coat) (Figure 2).
Figure 2.
Structure of solid lipid nanoparticles. Models of drug incorporation of solid lipid nanoparticles. (a) Homogeneous matrix model; (b) rich core model; (c) enriched roof model.
In general, the higher temperature of the aqueous medium facilitates more excellent solubility of the drug-containing them. Considering that in most studies, the temperature used to prepare the granules is typically at least 10° above the melting point of solid lipids, this operating condition facilitates a substantial amount of drug in phase. Then during cooling, drug solubility decreases, the aqueous phase is supersaturated, and in theory, the active molecules should migrate to the lipid substrate due to their lipophilic properties.
The outer shell or enriched shell structure can be obtained when solid lipids have a high melting point and a high crystallization temperature, which leads them to crystallize before the active molecule during the nanoemulsion system O/W hot cooling process housing the active molecule on the surface of the particle. This concentrated outer shell layer shows an explosion effect on drug release, so this model is unsuitable for prolonged drug release [34].
Conversely, suppose the use of fat has a low melting point or crystallization point. In that case, high temperature may remain as a supercooled liquid or in a metastatic crystalline form where more particles are present at high temperatures. Active elements can only be contained in rotation. Instead, it can promote nucleation and thus be encapsulated within the seed (core-shell structure) or uniformly dispersed throughout the grain structure, mainly when homogenization is used at high cold pressure.
The homogeneous matrix model can be obtained using the cold homogenization method and by incorporating lipophilic active molecules into the SLNs with the hot homogenization method. This solidified structure relies on its solid state to provide a uniform drug distribution in SLN [35, 36].
In the core-shell or enriched core structure, the concentration of the active molecule is close to the saturation point in lipid fusion; when this highly concentrated lipid is cooled, the solubility of the active molecule in lipid fusion is reduced, and it is deposited in the center of the SLN, forming a nucleus enriched with the active molecule. This type of structure leads to a sustained release profile [37, 38].
There are multiple methodologies for the preparation of SLN; the selected technique will depend on the physicochemical properties of the drug about the lipid matrix, the route of administration, among other parameters. The methods used for the preparation of SLN fall broadly into three main categories: (i) high-energy methods for dispersion of the lipid phase (such as high-pressure homogenization), (ii) low-energy methods where requires the precipitation of nanoparticles from homogeneous systems (such as microemulsions), and (iii) methods based on organic solvents.
4.1 High energy methods
High-energy-based methods are those that, in general, require the use of equipment capable of generating high shear forces, pressure distortions, or any other mechanism to achieve particle size reduction. In an emulsion, the mechanical energy required exceeds the interfacial energy by several orders of magnitude, thus requiring high energy to form submicron drops. The high-energy process uses intense mechanical force, resulting in large interfacial areas to form nanoscale emulsions [39]. The energy barrier for droplet fragmentation is highly dependent on the interfacial tension: the higher the interfacial tension, the more energy must be supplied to obtain tiny emulsion droplets. The interfacial energy and the curvature of the drop interface give rise to a pressure difference between the inside of the drop and the outside, called Laplace pressure. This pressure will act as a resistance to whatever stress is applied, so the reductions are expected to break approximately at the point when the applied stress equals or exceeds the Laplace pressure. The nanoemulsion droplets have a greater thickness due to the adsorption layer of the emulsifier concerning the radius of the droplet, which makes them more stable towards coalescence [26, 38]. In general, the high-energy process is followed by two steps: (i) the deformation and disruption of macro droplets into the smallest droplets; (ii) the adsorption of surfactant at its interface (to ensure steric stabilization) [40].
4.1.1 High-pressure homogenization (HPH)
Depending on the temperature used for the production of SLN, this technique can be classified into hot homogenization and cold homogenization. The advantage associated with this method is that SLNs are obtained with small particle sizes and high entrapment efficiency. High-pressure homogenization, the molten lipid is pumped through a narrow space with a 500–5000 bar pressure at high speed. Generally, 5–10% lipid content is used, but up to 40% lipid content has also been investigated. Two general approaches to HPH are hot homogenization and cold homogenization [41, 42, 43].
4.1.1.1 Hot homogenization
In the hot homogenization method, the procedure is carried out at temperatures above the melting temperature of the lipid. Here, the lipid and the drug are fused and combined with an aqueous surfactant at the same temperature. By using the high shear device, a hot pre-emulsion is formed. The hot colloidal emulsion droplets are recrystallized by cooling the emulsion to room temperature to generate the SLNs. In general, higher temperatures result in smaller particle sizes due to the decrease in the viscosity of the internal phase. However, high temperatures can also increase the rate of degradation of the drug and vehicle. In most cases, 3–5 homogenization cycles at 500–1500 bar are sufficient [41, 42]. Increasing the number of cycles or the homogenization pressure often increases particle size, inducing coalescence due to its high kinetic energy. Particle sizes < 500 nm can be obtained [42].
4.1.1.2 Cold homogenization
In this technique, the lipid melt containing the drug is cooled. Solid lipids are ground into lipid microparticles. These lipid microparticles are dispersed in a cold surfactant solution producing pre-excitation. This hypothetical process is then homogenized at room temperature or below; gravity is strong enough to reak lipid microparticles directly into SLNs [41, 42]. The particle sizes achieved by this technique are generally in the range of 50–1000 nm [41].
4.1.2 Ultrasonic/high-speed homogenization
This technique is based on the use of ultrasound waves which create cavitation phenomena that include the formation, growth, and implosive collapse of microbubbles/cavities in the medium; It consists of very high temperatures up to 5000 K and pressures up to 1000 bar. The particle size of the SLNs in this technique depends on the stirring speed, the emulsion time, and the cooling temperature, reaching heights less than 100 nm [44].
4.1.3 Supercritical fluid-based method
A supercritical fluid (SCF) increases the dissolving capacity of compounds, and fluid becomes a supercritical fluid when its temperature and pressure exceed its critical values. Supercritical fluid technology has the unique property of producing solids with a small size and irregular morphology. Supercritical fluids (SCF) have unique properties, such as high diffusivity, low viscosity, and high compressibility. Supercritical CO2 (SC-CO2) is the most common SCF because it is non-toxic, non-flammable, easy to obtain, and easily accessible. SLNs can be formulated by five main (SCF) methods: (i) rapid expansion of supercritical solutions (RESS), (ii) particles of gas-saturated solutions/suspensions (PGSS), (iii) supercritical fluid extraction of emulsions (SFEE) [45, 46].
4.2 Low energy methods
Low energy methods do not consume significant energy to achieve particle size reduction, and some even proceed spontaneously. These inferior energy methods are based on the properties of the system and its complex interfacial hydrodynamic mechanisms. The chemical energy released during emulsification is believed to occur as a consequence of the change in spontaneous curvature of surfactant molecules from negative to positive (o/w) or from positive to negative (w/o) [47].
Low energy techniques are classified into thermal or isothermal methods. Emulsion formation due to temperature-dependent changes in surfactant properties is typical of thermal processes, while emulsion formation due to continuous temperature changes is typical of the isothermal method. Spontaneous emulsion (self-emulsifying) and inverting emulsions are included among the isothermal techniques. Spontaneous emulsion involves adding an oily surfactant mixture to the water, whereas, for emulsion phase inversion, the emulsion is formed when water is added to the oily surfactant mixture. An emulsion is formed through the phase inversion temperature method when the temperature of the oil, water, and surfactant mixture rapidly drops below the phase inversion temperature under continuous mixing. These methods have the advantage of producing tiny droplets without specialized equipment. Therefore they are cost-effective and easy to use [48]. The following variables influence the spontaneity of the emulsification process: structure of the surfactant, concentration and initial location, composition of the oil phase, addition of co-surfactant and non-aqueous solvent, and salinity and temperature [49].
4.2.1 Microemulsion based method
The IUPAC defines the microemulsion as a dispersion made of water, oil, and surfactant (s) anisotropic and thermodynamically stable system with a dispersed domain diameter ranging from approximately 1–100 nm, generally 10–50 nm [50].
4.2.1.1 Hot microemulsion technique/microemulsion dilution technique
In this method, the microemulsion forms spontaneously due to the high surfactant-lipid ratio. This method is simple and includes some common steps. Initially, the fat is melted and mixed with a hot surfactant solution. Light shaking is carried out until a microemulsion is formed. In the second step, the hot microemulsion is dispersed in a large amount of cold water (2–10°C) with moderate stirring [51]. Temperature gradients facilitate the rapid crystallization of lipids and prevent aggregation. Due to the dilution step (1:25–1:50), achievable lipid contents are considerably lower than HPH-based formulations [52]. The particle size of the nanoparticles obtained varies from 50 to 800 nm [53].
4.2.1.2 Microemulsion cooling technique
The method consists of the preparation of an o/w microemulsion in which the emulsified wax melts at 37–55°C and the addition of water heated to the same temperature with minimal agitation to form a suspension homogenous milky white, after adding a specific amount of a pharmaceutically appropriate macromolecular surfactant to the water a stale and transparent microemulsion is produced in the form of a liquid matrix. This o/w microemulsion is further cooled to room temperature or 4°C to precipitate SLNs from it. This method is reproducible, simple, and easy to scale [54]. This method works at a moderate temperature, requires less time and obtains high drug entrapment. The particle size of the nanoparticles received varies from 50 to 300 nm [42, 54].
4.2.2 Double emulsion method
The double emulsion technique is one of the most used techniques to prepare nanoparticles encapsulated with hydrophilic active ingredients using stabilizers or surfactants. This method is also known as the multiple emulsion method, where it has three basic steps: (i) formation of the water-in-oil emulsion or reverse emulsion, (ii) addition of the W1/O emulsion in the aqueous surfactant solution to form a W1/O/W2 emulsion with continuous stirring (sonication or homogenization) and (iii) evaporation of the solvent or filtration of the multiple emulsion to form the nanoparticles [41].
4.2.3 Phase inversion method by temperature (PIT)
The temperature-induced phase inversion technique involves performing, using heating and cooling cycles, successive phase inversions of an O/W emulsion to a W/O emulsion until finally an emulsion is obtained. O/W correlation, where each inversion conducts to reduce the size of the droplet so that nanoparticles can be obtained with a final dilution step in cold water [36, 37]. This method brings SLNs of 30–100 nm in diameter.
4.2.4 Membrane contactor method
In this method, the lipid phase is pressed through the membrane’s pores and allows the formation of tiny droplets at the outlets of the pores, which are carried away by the circulating water. SLNs are obtained after cooling the preparation to room temperature [55]. This method can achieve particle sizes of 100–200 nm.
4.2.5 Coacervation technique
The coacervation method is based on the precipitation of free fatty acids from their micelles in the presence of a surfactant. In this process, a fatty acid salt is uniformly dispersed in the stabilizer solution. The mixture was heated to the Krafft point of the fatty acid salt and stirred continuously until a clear solution was obtained. Subsequently, an ethanolic solution of the API is slowly added under continuous stirring to get a single phase. Then, a coacervation agent or an acidifying solution is added to obtain the nanoparticle suspension [54]. The particle size of SLN depends on the concentration of the micellar solution and the degree of polymer used for stabilization and can produce a particle size of 260–500 nm [56].
In this technique, the lipid phase is heated above its melting point and dispersed in water using a low HLB surfactant. Again, an aqueous solution of surfactant with a high HLB content is added to the emulsion without thus forming; this double w/o/w emulsion is poured into cold water with gentle agitation to promote the formation of SLN [57].
4.3 Organic solvent approaches
4.3.1 Solvent evaporation emulsion method
The solvent evaporation emulsion (SEE) method has three basic steps for preparing nanoparticles. In step (I), lipid material is added to a known volume of organic solvent (immiscible in water) and suitably mixed to produce a clear homogeneous lipid solution. In step (II), the solution prepared above is added to the correct volume of a hot aqueous solution containing surfactant above the melting point of lipids to form a thick emulsion using a high-speed homogenizer. The nanoemulsion is then obtained in step (III) using a high-pressure homogenizer, which converts the coarse emulsion into a nanoemulsion due to the high pressure. Nanodispersion is formed after evaporation of the organic solvent since the lipid material will precipitate in the water. The lipids precipitated in an aqueous medium are separated by filtration through the sintered disk filter funnel. The nanoparticles prepared by this strategy are nano-sized, not flocculated (single entity), and have a high trapping efficiency [58]. This method can achieve 30–500 nm particle sizes.
4.3.2 Solvent emulsion and diffusion method
The method is based on the organic phase’s initial saturation and thermodynamic equilibrium with a stabilizer containing an aqueous phase. The drug is dissolved with the help of a homogenizer in the saturated solution formed; in the next step, an o/w emulsion is produced by dispersing it in an aqueous solution with an emulsifier. Diffusion of the solvent into water is facilitated by adding more water in an appropriate ratio to the emulsion under moderate magnetic stirring, which leads to nanoprecipitation and the formation of SLNs. Particles with an average diameter of 30,100 nm can be obtained with this technique [59].
In this technique, the lipid and the active ingredient are dissolved in a water-miscible solvent. The mixture is then dispersed in an aqueous solution of a surfactant with gentle mechanical agitation, producing a suspension of lipid nanoparticles; the solvent is subsequently removed. The particle size depends on the speed of the distribution process. Higher speed makes smaller particles. More lipophilic solvents give larger particles that can become a problem. The strategy offers advantages, for example, low temperatures, low cutting pressure, easy handling, and a fast production process without really advanced equipment (for example, high-weight homogenizer) [60]. The particle size of the nanoparticles obtained varies from 100 to 500 nm [53].
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.
In vitro release studies Fluorescence correlation spectroscopy (FCS)
Table 4.
Main parameters for the characterization of solid lipid nanoparticles.
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 non-uniformity 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 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].
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 high-energy 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:
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:
C=Ae−αt+Be–βtE3
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 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].
SLNs are the most studied lipid-based drug delivery systems, which can deliver drugs and nutrients for various routes of administration due to their biocompatibility, low toxicity, high loading capacity, slow-release rate, and high stability.
The physicochemical properties and stability of lipid nanoparticles depend on the composition of the lipid nanoparticle formulation. The lipid nature of these support systems is one of the key features that has attracted the interest of many researchers. Based on the organization of lipids and drugs in the particles, many structural models of SLNs have been described.
Lipid nanocarriers such as SLNs offer much more flexibility in drug loading, modulation of release, and improved performance in the production of final dosage forms such as creams, tablets, capsules, and injectables. The effort to develop alternative routes and treat other diseases with these systems must continue to expand their applications. Penetration through the gastrointestinal tract and BBB may be a new trend, and combining two therapeutically active agents in a single nanosystem is another consideration for future development.
In conclusion, solid lipid nanoparticles are a promising drug delivery system due to the non-toxicity aspect and a variety of drug-carrying capacity, along with the advantages of drug delivery through all routes of administration.
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Rosa-Alejandra Hernández-Esquivel, Gabriela Navarro-Tovar, Elvia Zárate-Hernández and Patricia Aguirre-Bañuelos
Submitted: 23 December 2021Reviewed: 07 January 2022Published: 02 November 2022
Open access peer-reviewed chapter
