A Review on Solid Lipid Nano Particles: Design, Characterization & Therapeutic Applications.

Nanotechnology serves as a foundational technology that engages with objects sized in the nanometer range. Future developments in this field are expected to unfold at different levels: materials, devices, and systems. Presently, the materials level of nanotechnology is the most sophisticated, demonstrating both advanced scientific knowledge and extensive commercial applications. (1) Nanomaterials (NPs) can be sized from zero to three dimensions, contingent upon their shape. The size of nanoparticles is crucial in determining the features of the material. (2)

Long before nanotechnology became a recognised field, people were inadvertently interacting with various nanosized materials and utilising nano-scale methods. In ancient Egypt, the common practice of dyeing hair black was believed for a long time to be based on plant-derived substances like henna. However, recent studies of hair samples from ancient Egyptian burial sites have shown that the dye was actually created from a mixture of lime, lead oxide, and water. This dyeing technique resulted in the formation of galenite (lead sulfide, PbS) nanoparticles. The ancient Egyptians were adept at making the dyeing paste react with sulfur (a component of hair keratin), leading to the production of small PbS nanoparticles that allowed for uniform and consistent dyeing.

One of the most notable examples of ancient nanotechnology is the Lycurgus Cup (fourth century CE). This Roman cup possesses extraordinary optical properties; it changes colour based on the angle of the light source. In natural light, the cup appears green, but when lit from within (for instance, by a candle), it takes on a red hue. Recent examinations of this cup revealed that it contains 50–100 nm Au and Ag nanoparticle which contribute to the cup's distinctive coloration through plasmon excitation of electrons. The historical use of nanotechnology does not end here; there is also evidence of its early application in Mesopotamia, Ancient India, and among the Maya. (3)

In nanotechnology, there are numerous forms, such as liposomes, Niosomes, transferosomes, and solid lipid nanoparticles. In these solid lipid nano particles are mostly used nowadays,In lipid and lipid-based drug delivery systems, phospholipids are vital components owing to their amphiphilic nature, biocompatibility, and multifunctionality. However, systems like liposomes, lipospheres, and microsimulation carriers have significant limitations, including complex production methods, low entrapment efficiency (% EE), and challenges in large-scale manufacturing. This has led to the emergence of the SLN delivery system. SLNs are generally spherical, with diameters between 50 and 1000 nm. The essential components of SLN formulations include lipids that are solid at room temperature, emulsifiers, and sometimes a combination of both, along with active pharmaceutical ingredients (APIs) and an appropriate solvent system Nanocarrier-based drug delivery systems can be categorised in various ways, depending on the route of administration and the degree of degradability. The routes of administration include nanoparticles for parenteral, oral, ocular, and topical use, as well as nanoparticles for protein peptide delivery. Furthermore, nanocarrier systems can be classified based on their degradability. An ideal nanoparticulate drug delivery system should possess the following characteristics:

1. Maximum drug bioavailability.
2. Targeting of specific tissues.
3. Controlled release kinetics.
4. Minimal immune response.
5. The ability to deliver traditionally challenging drugs such as lipophiles, amphiphiles, and biomolecules. [4]

Structure of solid lipid nanoparticles

SLNs consist of a solid lipid core at room temperature, forming a well-structured lipid matrix. This matrix effectively shields the active ingredient and regulates its release with improved quality. An outer surfactant layer stabilizes SLNs, allowing the active ingredient to be contained within the lipid core, at the surfactant interface, or distributed throughout the entire nanostructure. Developed in the 1990s, SLNs were designed to combat the rapid degradation and drug stability problems in liposomes, as well as the toxicity associated with polymeric nanoparticles from the use of volatile organic solvents.[5]                

Fig 1,2: solid lipid nano particle [6,7]

Types of solid lipid nano particle:

TYPE 1: Homogeneous matrix model

The homogeneous matrix model can be achieved through the cold homogenization method, which incorporates lipophilic active molecules into the SLNs using the hot homogenization technique.

TYPE 2: Drug-enriched shell model

In this technique, a solid lipid core is formed once the recrystallization temperature of the lipid is attained, leading to the drug concentrating in the still-liquid outer shell of the SLN due to a decrease in dispersion temperature.

TYPE 3: Drug-enriched core model

In this method, the active compound precipitates initially, resulting in the shell having a notably lower drug concentration. This results in a membrane-controlled release that follows Fick's law of diffusion. [8]

Fig 3: Types of solid lipid nanoparticles

Advantages:

• Capacity to integrate and transport a wide array of therapeutic agents.
• Superior physical and chemical stability.
• Ability to ensure controlled and sustained drug release.
• Decreased toxicity and adverse effects of the drugs being encapsulated.
• Enhanced biodistribution and bioavailability of the drugs within the encapsulation.
• Synthesis conducted under low toxicity conditions (in the absence of organic solvents)
• Economical and straightforward mass production.
• Lipid systems that are both biocompatible and biodegradable.
• Increased solubility and stability of the molecules incorporated.
• Improved permeability of drugs through the blood–brain barrier.
• Better drug permeability throughout the organism and retention in tissues.
• A broad range of administration routes, including oral, rectal, nasal, ocular, parenteral, and intravenous [9]

Disadvantages:

• restricted transdermal pharmaceutical delivery 
• High levels of drug loss 
• Inadequately controlled drug release mechanisms [10]

Methods of preparation:

1. Super critical fluid extraction method:

This method stands out as one of the most promising techniques for generating solid nanoparticles, based on the principle that lipid nanoparticles are formed from oil/water emulsions via supercritical fluid extraction (SCF).

The key advantage of this method over others is its operation at low temperatures (35°C) and the elimination of organic solvents in the nanoparticle fabrication process, which allows for solventless processing. Carbon dioxide (CO2) is commonly employed as the SCF, either on its own or with other solvents. The particles produced through this technique have smaller sizes and distributions, feature smooth surfaces, and are free-flowing, which supports the benefits of this method. Nonetheless, there are limitations to this approach, including costs, the low solvent strength of CO2, and the necessity for large quantities of CO2. SCF can act as a solvent, swelling and plasticizing agent, antisolvent, or solvent for polymerization in dispersed media during nanoparticle processing. The rapid expansion of supercritical CO2 solutions will yield solid lipid nanoparticles (SLNs). CO2 with a purity of 99.99% is an excellent solvent for preparing SLNs using this technique. Gas-saturated solutions (GSS), such as ammonia, chlorodifluoromethane (CHClF2), 1,1,1,2-tetrafluoroethane (CH2FCF3), and ethane, are the most effective SCFs and facilitate the melting of lipid materials, which then dissolve in the SCF under pressure along with the lipid melt and GSS. Spraying the saturated solution through an atomizer or nozzle causes it to expand and quickly release SCF, leaving fine dry lipid particles behind. The term “Supercritical fluid extraction of emulsions (SFEE)” refers to the method of producing lipid nanoparticles using emulsions through SCF technology.

The lipid component and the drug are dissolved in a suitable surfactant-containing organic solvent, such as chloroform, to create the organic phase. A high-pressure homogenizer is utilized to combine the organic solution with an aqueous solution that may also include a cosurfactant to form an o/w emulsion.

The supercritical fluid (maintained at a constant temperature and pressure) is currently supplied counter to the flow, while the o/w emulsion is delivered from one endpoint of the extraction unit (typically the top) at a steady flow rate. Continuous solvent extraction from o/w emulsions is being implemented. [11]

2. High-Pressure Homogenization:

 In the production of drug-loaded Nano-Structured Lipid Carriers (NLCs), cold high-pressure mixing and hot high-pressure mixing—commonly referred to as cold HPH and hot HPH—are the most widely used techniques. Hot HPH is defined as the preparation of medications using a blend of melted liquid lipids and solid lipids. The combination of lipids and hydrophobic drugs under elevated temperatures is subsequently homogenized with a high-pressure solvent evaporator, undergoing 3–5 cycles to ensure thorough mixing with a heated water-based solution that includes both hydrophilic surfactants (like Tween) and amphiphilic surfactants (such as lecithin).

This method forces the hydrophobic phase into a confined area within the homogenizer, leading to the creation of tiny dispersed particles in the submicron size range.HPH offers numerous advantages, such as higher processing temperatures that contribute to smaller particle sizes due to the reduced viscosity of the lipid phase. Nevertheless, employing hot HPH for the preparation of drug-loaded NLCs presents certain challenges. These challenges include the drug's sensitivity to heat, low entrapment efficiency, and a reduced drug-loading capacity, which may occur if drug molecules migrate into the hydrophilic phase during lipid crystallization Hot HPH has its drawbacks. To address this issue, we can utilize cold high-pressure homogenization (HPH) at temperatures and pressures typically below room temperature. In this approach, the lipid is melted while the drug is frozen using liquid nitrogen or dry ice. The exploration of cold high-pressure homogenization has opened new avenues for the advancement of nanostructured lipid carriers, enhancing the potential to encapsulate thermos-labile drugs effectively and improving drug-loading capacity. This process involves heating the drug-lipid mixture until it melts and then rapidly cooling it by submerging it in dry ice or liquid nitrogen. This swift cooling accelerates the crystallisation of solid lipids (SL). [12]

3.Microemulsion:

Microemulsion is one of the most widely recognised, frequently used, and popular methods for producing nanostructured lipid carriers (NLCs). It is preferred for its cost efficiency and versatility in managing both polar and non-polar drugs (Table 1). An emulsion created with melted fat oil or any other modifications should be prepared by incorporating it into surfactants and co-surfactants dissolved in a water solution containing these compounds, such as sodium dodecyl sulphate. The proportions of the components will determine whether the emulsion is oil-in-water (o/w) or water-in-oil (w/o). This ensures thorough mixing until small sizes, like microns, are achieved [41–43]. Nanostructured lipid carriers (NLCs) are formed by mixing this tiny emulsion with a cold aqueous mixture, known as the hydrophilic phase, at a ratio of 1:25 to 1:50 with gentle stirring; this process leads to a reduction in particle size.[12]

4. Double emulsion technique: -

 This method is extensively used for hydrophilic drugs, which are water-loving, and also for drugs that are sensitive to temperature. The double emulsion technique is applied to manufacture drug/biomolecule-loaded NLCs (like proteins and peptides)

 This technique produces a water-in-oil (w/o) emulsion by combining a hydrophilic drug dissolved in an aqueous medium with a lipid melt containing lipophilic surfactants. The next step involves adding this primary emulsion to an aqueous solution containing a hydrophilic solvent, creating a double emulsion that is water-in-oil-in-water (w/o/w). The Nanostructured Lipid Carriers (NLCs) are then separated from the dispersion by ultrafiltration and solvent evaporation. The double emulsion technique has an advantage over the single emulsion method because it does not require a molten lipid phase. Once these methods optimise the preparation of NLCs, their practical applications across various drug delivery systems become clear.[12]

5. Solvent Injection Method:

Another term for this is the "solvent displacement technique".The rapid and easy formation of lipids from their dissolution in a water-miscible solvent is achieved through solvent injection. This solution is promptly injected through a needle into an aqueous system that contains surfactants. This method consists of mixing lipids and pharmaceuticals in a water-soluble solvent, such as methanol, ethanol, acetone, isopropanol, or a combination of these solvents. The aqueous phase (water phase) is generally obtained by dissolving an emulsifier or a mixture in a water/buffer solution. Thus, the needle continuously stirs the aqueous phase while simultaneously introducing the organic phase rapidly under mechanical agitation. This technique provides the advantages of straightforward preparation and avoids the requirement for high heat, excessive shear stress, or complex equipment. However, its drawbacks include the risk of organic solvent residue, which can result in high particle concentrations.[12]

6. Solvent Evaporation method:

The lipophilic compound is dissolved in an organic solvent that does not mix with water (such as cyclohexane) and is emulsified in an aqueous phase. When the solvent evaporates, nanoparticles are formed by the precipitation of the lipid in the aqueous medium, yielding nanoparticles with a mean size of 25 nm. The emulsification of the solution in the aqueous phase was achieved through high-pressure homogenization. The organic solvent was removed from the emulsion by evaporation under reduced pressure (40–60 mbar).[13]

7. Solvent emulsification diffusion method

 This technique allows for the production of particles with average diameters ranging from 30-100 nm. The key advantage of this method is that it avoids heat generation during the preparation process. In this approach, lipids are generally dissolved in the organic phase within a water bath at 50 °C, and an acidic aqueous phase is utilised to modify the zeta potential, leading to the coacervation of SLN, which can then be easily separated by centrifugation. The SLN suspension is generated swiftly. The entire dispersed system can then be centrifuged and resuspended in distilled water.[13]

8. Spray drying:

An additional procedure to lyophilisation is available for the transformation of an aqueous dispersion into a drug. This method is more cost-efficient when compared to lyophilization. There is a risk of particle aggregation due to elevated temperatures, shear forces, and incomplete melting of the particles.[14]

9. Ultra Sonication:

SLN was also synthesised through high-speed stirring or sonication (Eldem et al., 1991). The tools required for this technique are standard in almost every laboratory. A key drawback of this method is the extensive particle size distribution that extends into the micrometre range, which is a leading cause of physical instability (Svilenov and Tzachev, 2009). Concerns such as particle growth during storage and the risk of metal decay are pressing issues in this approach. After extensive studies and thorough research, it has been confirmed that high-speed stirring and ultrasonication, when used together at high temperatures, produce a reliable formulation.[14]

10. Phase Inversion Temperature

Phase Inversion Temperature (PIT) Methodology. This methodology is founded on the capability of non-ionic surfactants to modify their affinity for water and oil based on temperature fluctuations. In this process, the oil and surfactant blend is melted and then introduced to water while being stirred continuously. The system is maintained at a heated state until it reaches the cloud point, followed by a rapid cooling phase. During the heating phase, the oil/water emulsion shifts to a water/oil configuration, and upon cooling, an oil/water emulsion is formed once more. With each phase inversion, the size of the particles diminishes, enabling the production of nanoparticles at the end of the procedure.[15]

Fig 4, 5, 6,7&8. Figures explaining the process of Supercritical fluid extraction, Solvent emulsification diffusion method, double emulsion, hot and cold HPH, microemulsion, solvent evaporation, ultrasonication, solvent injection, and phase inversion temperature (PIT). [15,16]

DRUG LOADING AND RELEASE FROM SLN

The particle size and the type of drug entrapment model for SLNs play a crucial role in drug release, in line with the established theory of drug release from nanoparticles. Elements such as the drug solution and its interaction with the lipid matrix can affect the release of drugs. Temperature changes can influence the release profile of SLNs in response to both internal and external factors. Chen et al. studied the pH-sensitive release profile of cholesterol-PEG-coated SLNs loaded with doxorubicin and found that the drug released more quickly at pH 4.7 than at pH 7.4.

In general, SLNs displayed burst release. As the size of the particles increases, the burst release of the drug may reduce, resulting in a more extended release. Using tetracaine, etomidate, and prednisolone as model drugs, zur Mühllen et al. found that SLNs containing tetracaine and etomidate exhibited a burst drug release (100% release in under 1 minute) due to their significant surface area and drug enhancement in the outer shell. Conversely, prednisolone-loaded SLNs were shown to have a 5-week extended release. Burst (83.8%) and regulated releases (37.1%) were achieved due to the specific chemical activity of the lipid matrix, such as that of cholesterol and compritol.

According to Olbrich and Muller, a lipid interface is required for the activation of the enzymes that decompose the lipid matrix. It is recommended to modify the surface of SLNs with a hydrophilic carrier (such as PEG) to prevent lipase enzymes from easily recognizing them, thereby altering their release and enhancing their stability. Appropriate steric stabilizers and other surfactants should also be fine-tuned.

A recent review by Savla et al. indicates that drugs with a high melting point (not numerically defined) and a Log P value of 2 are typically poor candidates for lipid systems, especially for highly lipophilic drugs. [17]

Types of Materials used in Solid lipid Nanoparticles;

Table 1: Types of materials used in the preparation of solid lipid nanoparticles. [18,19]

Characterisation of SLNPS:

1. Zeta potential:

Zeta potential measurement can be performed using a zeta potential analyser or zetasizer. Before measurement, SLN dispersions are diluted 50-fold with the original dispersion preparation medium, determining size and measuring zeta potential(Luo et al., 2006). A higher zeta potential value may result in the deaggregation of particles if there are no other complicating factors, such as steric stabilisers or hydrophilic surface appendages. Zeta potential measurements provide insights into the storage stability of colloidal dispersions.[20]

2. FTIR:

An FT-IR spectroscopic analysis was conducted to investigate the interaction between drug excipients. The distinct peaks of the drug within the IR spectrum of the mixture of drug and drug excipients were identified by absorption. Therefore, FTIR Spectroscopy was utilised to gather conformational data regarding the lipid molecules, and it was employed to explore the interactions among lipid, drug, and excipients, confirming that no significant interactions occurred between them, allowing the drug to remain unchanged in the formulation. [21]

3. SEM:

The SLNs suspension of SA-SLNs-SE and SA-SLNs-DE was treated with gold spray, and the surface morphology was observed using a scanning electron microscope (SEM) (S4800, Japan). The microscope operated at a voltage of 5 kV during the observation.[22]

4. TEM:

The morphology of the optimised SLNP was analysed using transmission electron microscopy (TEM) (JEM-2100F, Japan). The SLNs suspension was placed on a carbon-coated copper grid, followed by a drop of 2% phosphotungstic acid on the SLNs layer. After a 5-minute staining, excess dye was removed from the edges using filter paper. The sample was dried at room temperature for about half an hour and was then prepared for TEM investigation at 100 kV. [22]

5.Thermal behaviour:

Differential scanning calorimetric (DSC) analysis was executed to validate the drug-lipid association in nanoparticulate formulations and the crystallinity of the drug with the Mettler TASTARe system featuring a DSC 822e cell (Mettler Toledo, US). Samples comprising 40 mg of SLNs were weighed with precision in aluminium pans. DSC scans were recorded at a heating and cooling rate of 5º/min. The DSC analysis was performed on plain drug, blank SLNs, and drug-loaded SLNs. The SLNs were dried under vacuum at room temperature to remove moisture prior to the DSC study.

Differential scanning calorimetric (DSC) analysis was executed to validate the drug-lipid association in nanoparticulate formulations and the crystallinity of the drug with the Mettler TA STARe system featuring a DSC 822e cell (Mettler Toledo, US). Samples comprising 40 mg of SLNs were weighed with precision in aluminium pans. DSC scans were recorded at a heating and cooling rate of 5º/min. The DSC analysis was performed on plain drug, blank SLNs, and drug-loaded SLNs. The SLNs were dried under vacuum at room temperature to remove moisture prior to the DSC studies.[23]

6.Crystallinity analysis:

X-ray powder diffraction (pXRD) patterns were recorded on a Philips PW 17291 powder X-ray diffractometer (Philips, the Netherlands) utilizing Ni-filtered Cu-K radiation, with a voltage of 40 kV and a current of 25 mA. The scanning rate was maintained at 1° min−1 within the 10?40° 2θ range. pXRD diffraction patterns were documented for the plain drug, bulk lipids, and drug-loaded SLNs. The SLNs underwent vacuum drying at room temperature to remove water before the pXRD studies. [23]

7.In Vitro Drug Release

The drug release was analysed in 1× simulated gastric fluid (SGS) and 1× phosphate buffer saline (PBS) through the dialysis-bag method. A total of 2 mg of drug -SLN was placed in 1 mL of PBS within the dialysis bag and kept at 37 ± 1 ?C under magnetic stirring at 100 rpm in a 10 mL tube containing either SGS or PBS. At regular time intervals, aliquots of the dialysate were withdrawn, and the same volume of fresh medium was replenished. The samples were collected at various time points (i.e., 0, 10, 20, and 30 minutes, as well as 1, 2, 3, 4, 6, 7, 8, 9, 10, and 12 hours) for kinetics analysis, filtered through a 0.2 µm filter, and analysed by HPLC  or UV at the required wave lengh, All experiments were conducted in triplicate, independently.[24]

8. Entrapment Efficiency:

Entrapment Efficiency: The efficiency of drug entrapment is determined by measuring the concentration of free drug in the dispersion medium.Ultracentrifugation was executed using Centrisart, which comprises a filter membrane (molecular weight cutoff 20,000 Da) at the bottom of the sample recovery chamber. The SLNs, along with the encapsulated drug, stay in the outer chamber while the aqueous phase flows into the sample recovery chamber. The concentration of the drug in the aqueous phase is assessed using HPLC or a UV spectrophotometer. [25]

% Entrapment efficiency = [(Initial drug weight–weight of free drug) / Weight of initial drug] × 100%

9. Stability Studies:

A stability study was performed on best formulation, which exhibits high entrapment efficiency. The formulation was stored at two distinct temperatures, 4 °C and 25±2 °C, and the drug content was assessed every 15 days to detect any variations in the entrapment efficiency of the SLNs [21].

Patents of Solid Lipid Nanoparticles:

Table 2: patents of solid lipid nanoparticles [26]