2.1. High-Pressure Homogenization Method

High-pressure homogenization (HPH) has been widely used to prepare nanoemulsions, SLNs, and NLCs. The technique is based on the reduction of droplet and particle size under extreme pressure conditions ^[12]. It provides an effective, reliable means of preparing SLNs and NLCs on a large-scale. For example, this method was used to prepare stavudine-loaded SLNs at batch sizes of up to 60 kg ^[13] and coenzyme Q10 loaded-NLCs at a throughput of 25 kg/h ^[14]. Initially, coarse lipid particles are prepared and then forced through a homogenizer with a narrow gap of few microns at high pressure ^[8]. The pressures (usually 100–2000 bar) accelerate the liquid mixtures to a high velocity of >1000 km/h, which generates high shear stresses and cavitational forces that reduce droplet sizes to nano dimensions ^[1]. This homogenization process is cycled until the desired droplet size and uniformity is obtained. The advantages of HPH are short production times, ease of production, organic solvent-free operation, and scale-up feasibility ^[14].

Two approaches are used to produce SLNs and NLCs using HPH, which are hot and cold homogenization. For hot HPH, drugs and molten lipids are mixed at temperatures typically 5–10 °C higher than the solid lipid melting point; drugs are either dissolved or homogeneously dispersed in the molten lipids ^[15]. Generally, the concentration of lipids in resultant SLNs and NLCs dispersion is 5–10% (w/v). Separately, an aqueous phase containing surfactants is preheated to the same temperature as the lipid melt and then added under constant stirring to produce a hot pre-emulsion. The resultant pre-emulsion is homogenized using a piston-gap homogenizer at the same temperature ^[16]. Generally, desired SLNs and NLCs can be obtained after 3–5 homogenization cycles at 500–1500 bars. However, increasing cycle numbers and homogenization pressure may increase particle size (PS) due to particle coalescence under highly kinetic conditions ^[17]. After homogenization, the nanoemulsions are cooled, which results in lipid crystallization and the formations of SLNs and NLCs. On the other hand, hot HPH has its limitations, which include temperature-induced drug degradation and drug loss to the aqueous phase during homogenization ^[18]. Therefore, the technique is unsuitable for heat-sensitive or hydrophilic drugs. The temperature sensitivity issue can be overcome by cold HPH (as discussed below), whereas drug loss is usually minimized using covalently bonded lipid-drug conjugates ^[19]. 

Cold HPH was developed to overcome some of the limitations of hot HPH. For cold HPH, after dissolving/dispersing drugs in molten lipids, mixtures are rapidly cooled using liquid nitrogen or dry ice. This high cooling rate provides homogeneous dispersions of drugs in lipid matrices ^[1]. The lipid-drug mixtures are then pulverized using a ball mill or a mortar to a PS of 50–100 µm. The lipid microparticles are suspended in cold aqueous solutions containing surfactants and then homogenized at the cold condition (e.g., 0–4 °C) usually over 5–10 cycles at 500 bars ^[20]. Microparticle suspensions can also be prepared in ways that avoid the laborious pulverization step. For example, a solvent diffusion method was used to prepare a coarse suspension, which was subsequently subjected to cold HPH at 0–4 °C ^[21][22], which minimized drug degradation associated with hot HPH ^[18]. In addition, cold HPH is suitable for water-soluble drugs as it suppresses drug migration from the lipid phase to the aqueous phase ^[1]. In addition, several modifications can be used to improve EE and DL values further. For example, pH values of the aqueous phase can be adjusted to minimize losses of drugs with pH-dependent solubility to the aqueous phase. Drug-lipid conjugates can also be used. To prepare decitabine-loaded SLNs, decitabine-stearic acid conjugates were ground into a fine powder, dispersed in a cold aqueous solution containing surfactants, and subjected to 10–14 cycles of cold HPH at a pressure of 1000 bar, at which an EE of up to 68.9% was obtained ^[23]. On the downside, it should be noted that cold HPH produces larger particles with a broader particle size distribution than hot HPH ^[1].

2.2. High-Speed Stirring and Ultra-Sonication Methods

High-speed stirring (high-shear homogenization) and ultra-sonication are widely-used dispersing techniques. High-speed stirring is one of the simplest and most cost-effective ways of producing SLNs and NLCs ^[18]. According to this method, lipids are first melted at high temperatures (5–10 °C higher than the melting point of solid lipids), and drugs are dissolved or dispersed homogeneously in the molten lipids. An aqueous phase containing surfactants (at the same temperature) is then added to the drug-lipid melt, and the mixture is homogeneously dispersed using a high-shear mixer. A hot oil/water (o/w) emulsion is formed due to the shear of intense turbulent eddies. SLNs and NLCs are formed by cooling these dispersions ^[24]. This high-speed stirring is usually followed by ultra-sonication, which breaks droplets based on the formation, growth, and implosive collapse of bubbles ^[25]. When ultra-sonication is performed without the high-shear mixing stage, SLNs and NLCs produced have a broad distribution ^[26], presumably because sonication energy is not transferred equally in the batch. High-speed stirring and ultra-sonication have been widely used in combination to achieve SLNs and NLCs dispersions with narrow particle distributions ^[27]. Both high-speed stirring and ultra-sonication are easy to handle and can be used widely without organic solvents ^[28]. However, both suffer from the disadvantage that drugs are exposed to high temperatures for extended times ^[29]. Moreover, the size distributions of SLNs and NLCs produced by high-speed stirring are broad and particles are micro-sized. The disadvantage of ultra-sonication is that products are contaminated with metals originating from sonicator probes ^[1]. Bath sonication provides a means of circumventing this problem but must be combined with other follow-on procedures to reduce PS and PDI values. Furthermore, using high-speed stirring and/or ultra-sonication methods require high surfactant, whereas total lipid concentrations are low ^[30].

2.3. Microemulsion Method

This method was first developed in the early 1990s and has since been investigated by several researchers ^[31]. The method involves diluting a microemulsion in a cold aqueous solution, which results in the formation of nanoemulsion and the subsequent formation of SLNs and NLCs by lipid precipitation. Briefly, a drug is dissolved in molten lipids at a temperature above the lipids melting point, and then an aqueous phase containing water and surfactant (pre-heated to the same temperature) is added under mild stirring to form a transparent and thermodynamically stable microemulsion ^[32]. The microemulsion is then poured into a cold aqueous solution (2–10 °C) under gentle mechanical mixing ^[18]. Typically, the volume of the cold aqueous phase is 25 to 50 times greater than that of the hot emulsion ^[1]. Upon dilution, a nanoemulsion is formed and lipids immediately crystallize to form SLNs or NLCs ^[33]. The microemulsion method is simple and reproducible and can be scaled up. For large-scale production, the microemulsion can be prepared in a large temperature-controlled tank and pumped into another tank containing cold water for lipid precipitation ^[18]. In addition, this method is solvent-free. Its limitations are a large amount of water required to dilute microemulsions and high surfactant usage ^[34]. Excess water can be removed by lyophilization or ultra-filtration to concentrate SLNs and NLCs dispersions ^[18].

2.4. Solvent Emulsification-Diffusion Method

The solvent emulsification-diffusion method is mainly used to produce polymeric nano-carriers. In 2003, Trotta et al. first used this technique to prepare SLNs and NLCs ^[35]. This method is generally performed using organic solvents that are partially miscible with water, such as methyl acetate, ethyl acetate, isopropyl acetate, benzyl alcohol, and butyl lactate. Initially, the organic solvent and water are mutually saturated with each other to obtain the initial thermodynamic equilibrium of both phases. Lipids and drugs are dissolved in the water-saturated solvent, which is then emulsified in the aqueous phase (solvent-saturated water-containing stabilizer) under stirring to form an o/w emulsion. The emulsion is diluted with water (volume ratio from 1:5 to 1:10) to allow diffusion of the solvent into the continuous phase. SLNs and NLCs are formed spontaneously due to lipid precipitation and the solvent is then eliminated by lyophilization or vacuum distillation ^[36]. This method can avoid exposing drugs to high temperatures and physical stress like those associated with high-speed stirring or high-pressure homogenization. In addition, it can be easily scaled up. Furthermore, the technique can be applied to hydrophilic and hydrophobic drugs. Different drugs and biomolecules, such as insulin, tretinoin, and cyclosporine have been encapsulated in SLNs and NLCs using this technique. However, like the microemulsion method, the solvent emulsification-diffusion method is associated with substantial dilution of SLNs and NLCs dispersions and requires a purification process to remove residual organic solvent ^[18].

2.5. Solvent Emulsification-Evaporation Method

Unlike the emulsification-diffusion method, water-immiscible organic solvents (e.g., chloroform, cyclohexane, dichloromethane, and toluene) are used to prepare SLNs and NLCs using the solvent emulsification-evaporation method ^[37]. Briefly, drugs and lipids are dissolved in a solvent or a solvent mix and then emulsified in an aqueous phase to form nanodispersions. Thereafter, the organic solvent is evaporated by mechanical stirring or in a rotary evaporator. SLNs and NLCs are formed due to lipid precipitation after solvent evaporation ^[38]. The concentration of lipids in the organic phase has a considerable effect on mean PSs of SLNs and NLCs prepared using the solvent emulsification-evaporation method. Small SLNs and NLCs are obtained at a low lipid concentration ^[1]. The main advantage of this method is avoidance of drug exposure to high temperatures, and thus, it is useful for encapsulating highly thermo-labile drugs. Furthermore, SLNs and NLCs produced using this method and the solvent emulsification-diffusion method have PSs of around 100 nm and narrow size distributions. The limitations of the solvent emulsification-evaporation method are that it requires toxic organic solvents and the suspension produced is dilute and requires further evaporation or ultra-filtration ^[29]. Both the solvent emulsification-diffusion and solvent emulsification-evaporation methods involve the use of organic solvents, which means additional solvent removal steps are needed. Furthermore, in vitro and in vivo risk assessments on the SLNs and NLCs produced are also required. The residual solvent levels should be below specified values, at which no adverse effect is observed ^[39].

2.6. Double Emulsion Method

The double emulsion method provides a means of producing SLNs and NLCs of hydrophilic drugs and biomolecules (e.g., peptides and proteins) ^[40]. Particularly, it has been widely used to incorporate insulin into the matrices of SLNs and NLCs for oral delivery ^[41]. According to this method, a drug and a stabilizer are dissolved in an aqueous solution and then emulsified in a water-immiscible organic phase containing lipids ^[42] or in solvent-free molten lipids ^[43][44]. These primary emulsions are dispersed in an aqueous phase containing a hydrophilic emulsifier to form water/oil/water (w/o/w) emulsion. After solvent evaporation, SLNs and NLCs dispersions are obtained due to lipid precipitation ^[45]. Notably, for this method, the stabilizer essentially prevents drug partitioning to the external water phase during the solvent evaporation. The advantage of the double emulsion method is that it does not require molten lipid. However, EE and DL values of SLNs and NLCs are low due to the leaching of the hydrophilic drugs to the outer aqueous phase. EEs of SLNs and NLCs produced using this technique may range from 5.2–40.3% for insulin ^[45] or relatively higher (63%) for sulforhodamine 101 ^[44]. In some cases, EEs can also reach 80% for diethyldithiocarbamate ^[43] and 90% for polymyxin B sulfate ^[46]. In addition, the SLNs and NLCs prepared using this method have relatively large PSs (up to micro sizes) ^[18].

2.7. Phase Inversion Temperature (PIT) Method

The PIT method has been used to produce nanoemulsions, SLNs, and NLCs ^[47]. The technique is based on the temperature-induced inversions of w/o to o/w emulsions and vice versa. The method requires the use of non-ionic polyoxyethylated surfactants with temperature-dependent properties. The hydrophilic-lipophilic balance (HLB) value of these surfactants is high at low temperatures because their hydrophilic groups are highly hydrated. When the temperature is increased, dehydration of the ethoxy group occurs, which increases surfactant lipophilicity and decreases HLB values of the surfactants ^[48]. PIT is defined as the temperature at which the affinities of the surfactants for aqueous and lipid phases are equal ^[49]. At temperatures > PIT, the surfactants favor the formation of w/o emulsions, whereas, at temperatures < PIT, they turn to form o/w emulsions ^[50]. To produce SLNs and NLCs, oil, water, and surfactant are first heated to a temperature > PIT under stirring to form w/o emulsions. Subsequently, they are rapidly cooled with continuous stirring, which promotes the breakdown of w/o microemulsions and induces the formation of o/w nanoemulsions. SLNs and NLCs are formed when lipids are precipitated at low temperatures ^[51]. SLNs and NLCs prepared using this method have been reported to have small PSs, narrow size distribution, and excellent stabilities ^[52]. This method requires little energy input and does not require organic solvents. However, it involves low stabilities of the nanoemulsion formed, and sometimes requires several temperature cycles (e.g., three cycles between 60 and 90 °C) ^[53].

2.8. Membrane Contactor Method

In this method, a specific membrane contactor is used for producing SLNs and NLCs. Briefly, a lipid phase is pressed through the pores of a membrane while the temperature is maintained above the solid lipid’s melting point. This step results in the formation of small droplets. At the same time, an aqueous phase containing surfactants circulates over the other side of the membrane inside a module. It flows tangentially to the membrane surface and sweeps away droplets formed at pore outlets. SLNs and NLCs are formed by allowing the hot emulsion to cool down to room temperature ^[54]. The effects of formulation and process parameters, including the membrane pore size, lipid phase pressure, flow velocity of the aqueous phase, and temperatures of the aqueous and lipid phases on SLNs and NLCs size are intensively investigated. PS can be adjusted by changing lipid phase flux through membranes. This method has been used to prepare vitamin E-loaded liposomes, micelles, nanoemulsions, and SLNs for pulmonary drug delivery ^[55]. This method is feasible for scaling-up as a membrane with a pore size of 0.1 μm resulted in high fluxes ^[56]. However, the limitations of this method are that it requires a sophisticated system and membranes are prone to clogging.

2.9. Supercritical Fluid-Based Methods

Several SLNs and NLCs production methods involved the use of supercritical fluids like supercritical CO₂. For the supercritical fluid extraction of emulsions (SFEE) method, an o/w emulsion is prepared beforehand, followed by supercritical fluid extraction of the organic solvent. Typically, the emulsion is added to an extraction column from the top, and supercritical CO₂ is introduced in a counter-current manner from the bottom. SFEE has a much higher solvent extraction efficiency than other methods that use evaporation, diffusion, and dilution. The solvent is quickly and completely removed, and this leads to lipid precipitation. Furthermore, the produced SLNs and NLCs have uniform particle size distribution ^[57]. The o/w emulsions are prepared using water-immiscible solvents ^[57] and water-partially miscible solvents ^[58]. 

Supercritical CO₂ is also used in a method called supercritical assisted injection in a liquid antisolvent (SAILA). This method is similar to the solvent injection method, which is discussed below. In brief, lipids and drugs are dissolved in a water-miscible organic solvent, mixed with supercritical CO₂ and injected into an aqueous phase containing surfactant. The mixing of the two fluids results in a rapid supersaturation and consequent precipitation of SLNs or NLCs ^[59]. Supercritical fluid-based methods usually result in uniform particle size distributions and their solvent extraction efficiencies are higher than those of conventional extraction methods. However, they are expensive and involve the use of organic solvents ^[58].

2.10. Coacervation Method

Coacervation has been widely used to produce polymeric nanoparticles and was first used by Battaglia et al. to prepare SLNs in 2010 ^[60]. This method is based on the acidification-induced precipitation of the alkaline salts of fatty acids. The lipids used are in alkaline salt forms (e.g., sodium stearate), which are dispersed in an aqueous solution of a polymeric stabilizer, such as PVA or HMPC ^[61]. Drugs can be dissolved in the lipid phase ^[62] or loaded into blank SLNs in later steps ^[63]. To load drugs into the lipid phase, drugs are solubilized in ethanol and dissolved in the lipid. The mixture is heated to a temperature above its Krafft point when a clear micellar solution of the lipid alkaline salts is formed. An acidifying solution (coacervating solution) is then added dropwise to this solution, which causes the lipids to precipitate. The obtained suspension is then cooled in a water bath with stirring at 300 rpm until a temperature of 15 °C is reached then cooled in a water bath with stirring to complete the precipitation of SLNs or NLCs ^[64]. The coacervation method provides a simple solvent-free means of preparing SLNs and NLCs without sophisticated instruments. However, it can only be used on lipids that form alkaline salts, such as fatty acids, and is not suitable for pH-sensitive drugs ^[64].

2.11. Solvent Injection Method

The solvent injection method used for preparing SLNs and NLCs was first reported by Schubert et al. in 2003 ^[65]. According to this method, lipids and drugs are dissolved in a water-miscible solvent (e.g., methanol, ethanol, isopropanol, or acetone) or a water-miscible solvent mixture. The aqueous phase is usually prepared by adding an emulsifier or an emulsifier mixture to water or a buffer solution. The organic phase is then quickly injected into the aqueous phase under continuous mechanical stirring using a needle ^[18]. The basic principles of this method and the solvent emulsification-diffusion method are similar. Following injection, two principal mechanisms occur simultaneously and aid each other to form SLNs and NLC. First, the solvent diffuses out of the droplets into the aqueous phase, which results in a droplet size reduction. As a consequence, lipid concentration within the droplets increases, which leads to the formation of local supersaturated regions stabilized by emulsifiers in the aqueous phase ^[65]. Second, the emulsifiers reduce the interfacial tension between water and solvent, and this leads to the formation of small solvent-lipid droplets at the injection site. Due to the interfacial pulsation and turbulence during solvent diffusion, those droplets are broken into smaller droplets with essentially the same lipid concentrations ^[65]. The free energy released when the solvent is redistributed to its equilibrium state provides the energy required for droplet division ^[65]. Therefore, in the solvent injection method, solvent diffusion results in the formation of tiny droplets and lipid precipitation. Emulsifiers play an important role in the determination of PSs and size distributions. Consequently, SLNs and NLCs are formed and stabilized by the emulsifier. Notably, the diffusion rate of organic solvent into the aqueous phase is considered to be one of the most critical factors affecting PSs and size distributions ^[65][66].

This method has been modified in some studies, including a micro-channel with a cross-shaped junction ^[67] or a co-flowing micro-channel system ^[68]. The micro-channel with a cross-shaped junction consists of a microchannel module, two precision syringe pumps for supplying an aqueous phase and a solution of lipid in solvent, a digital inversion microscope, and a stirred unit to collect the SLNs suspension. The process was carried out as follows: an organic solution (Softisan 100 in acetone, viscosity 0.35 mPa) was fed into the main microchannel, and an aqueous phase (0.5% poloxamer 188, viscosity 1.46 mPa) was pumped into the inlets of the two branch channels at an equal flow rate. A digital inversion microscope system was used to capture images of flow field behaviors in microchannels. The produced SLNs had PSs of 100–200 nm ^[67]. In the co-flowing micro-channel system, two precision syringe pumps were also used to supply the aqueous and lipid-solvent phase. The inner capillary had inner and external diameters of 110 and 490 μm, respectively, whereas these values of the outer capillary were 650 and 7000 μm, respectively. An organic solution (Softisan 100 in acetone) was fed into the inner capillary, and simultaneously, an aqueous phase (poloxamer 188) was pumped into the outer capillary at the same flow rate. The produced SLNs had small PSs (<250 nm) and narrow size distribution (PDIs <0.26) ^[68]. Another modification of the solvent injection method is the microfluidic rapid ethanol dilution method using baffle devices. This method was used as a post-treatment and provided precise size control of SLNs and NLCs prepared using the solvent injection method. For the SLNs and NLCs preparation step, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in ethanol and saline were introduced into the baffle device. Post-treatment was performed on-device and involved diluting the SLNs and NLCs suspensions (with 25% v/v ethanol) to 1%. This post-treatment was applied to prepare siRNA-loaded NLCs with a small PS (33 nm) and a high EE (>90%). The formulation specifically knocked down the plasma coagulation factor VII ^[69]. Table 1 summarizes the mechanisms, advantages, and disadvantages of different SLNs and NLCs preparation methods.