6.1. Encapsulation techniques
The beneficial effects of therapeutic agents discussed earlier will only be realized if they can reach intended site of action within human body in a bioactive form. There are a number of issues currently limiting the bioavailability and bioactivity of therapeutic agents deemed effective against atherosclerosis. Initially, it is important to be able to formulate a delivery vehicle that contains a sufficiently high dose of the therapeutic in a chemically stable and bioavailable form. For oral administration, this formulation may be in the form of a pill, capsule, fluid, or functional food. It is important that this formulation is designed to inhibit any chemical degradation of the therapeutic agent during production, transport and storage but that it then releases the therapeutic agent in a bioavailable form in the human gastrointestinal tract. The design of an effective formulation is highly dependent on the nature of the therapeutic agent and the delivery format and must be established on a case-by-case basis (McClements, 2018). Some of the most important factors to take into account when designing an efficacious formulation are the polarity (LogP), water solubility, melting point, charge, and chemical reactivity of the therapeutic agent.
Under physiological conditions, resveratrol is a strongly hydrophobic molecule (LogP = 3.4) with a low water solubility (0.14 mg/L) and poor chemical stability (especially under alkaline conditions) (Zhou, Zheng & McClements, 2021a), which reduces its bioavailability and bioactivity and therefore limits its application as a therapeutic agent in the pharmaceutical industry (De La Lastra & Villegas, 2007; Erlank, Elmann, Kohen & Kanner, 2011; Salehi et al., 2018). Similarly, melatonin is a modestly hydrophobic molecule (LogP=1.15) with a relatively low water solubility (1.0 mg/mL) (chemicalize.com). Melatonin has also been reported to chemically degrade when dispersed in aqueous solutions, with the rate of degradation increasing with increasing pH, temperature, and light exposure (Daya, Walker, Glass & Anoopkumar-Dukie, 2001; Pranil, Moongngarm & Loypimai, 2020). The limited water solubility and chemical stability of melatonin reduces its bioavailability and bioactivity, which again limit its efficacy as an effective therapeutic agent (Molska, Nyman, Sofias, Kristiansen, Hak & Widerøe, 2020). For these reasons, there has been considerable interest in the utilization of nanotechnology-based encapsulation methods to improve the efficacy of resveratrol and melatonin as therapeutic agents in drugs, supplements, and functional foods (Chuffa et al., 2021; Schaffazick, Pohlmann & Guterres, 2007; Zhou, Zheng & McClements, 2021a; Zhou, Zheng & McClements, 2021b).
Nanoenabled-encapsulation typically involves trapping the therapeutic agent within small (colloidal) particles, which typically have dimensions somewhere between around 10 and 1000 nm (McClements, 2020a; McClements, 2020b). Having said this, larger particles are sometimes utilized for certain applications. The colloidal particles may be solid, semi-solid, or liquid and may be fabricated from a range of different natural and synthetic ingredients, including proteins, polysaccharides, lipids, phospholipids, surfactants, and synthetic polymers. In general, these particles may vary in their size, shape, composition, charge, physical state, internal structure, and aggregation state, which means that their properties can be tailored for specific applications. It should be noted that these properties may change once the particles are incorporated into a formulation or after they enter the human gut, which needs to be taken into account for drug design purpose.
The entrapped ingredients (in this case therapeutic agents) are often referred to as the “core material” whereas the surrounding matrix is referred to as the “encapsulant” or “shell material” (Nedovic, Kalusevic, Manojlovic, Levic & Bugarski, 2011). Encapsulation has been shown to enhance the dispersibility of therapeutic agents in aqueous solutions, to protect them against chemical degradation by environmental factors, and to promote their absorption in gastrointestinal tract (Davidov-Pardo & McClements, 2014). Emulsions and nanoemulsions, microemulsions, liposomes, and cyclodextrins are among the most commonly used encapsulation technologies for this purpose.
Oil-in-water emulsions and nanoemulsions are composed of oil, water, and emulsifiers, which exist as numerous small emulsifier-coated spherical oil droplets suspended in water (McClements & Rao, 2011). By definition, droplets in nanoemulsions present diameters below 200 nm whereas those in emulsions have diameters above this value. The smaller dimensions of oil droplets in nanoemulsions offer benefits for certain encapsulation applications, including increased optical clarity, greater storage stability, and higher bioavailability of therapeutic agents. Emulsions and nanoemulsions can be used to encapsulate lipophilic bioactive compounds such as resveratrol and melatonin within their hydrophobic cores (Donsì, Sessa, Mediouni, Mgaidi & Ferrari, 2011; Rondanelli et al., 2012). Studies have shown the encapsulating melatonin within oil-in-water nanoemulsions significantly increases its physicochemical stability and solubility (Molska, Nyman, Sofias, Kristiansen, Hak & Widerøe, 2020). Nevertheless, it is important to carefully select the oil phase of emulsions and nanoemulsions so that it can inhibit any chemical degradation of the therapeutic agents during storage, as well as to ensure that it promotes their bioavailability after ingestion. For instance, it has been shown that long chain triglycerides are more effective than medium chain triglycerides at increasing the bioavailability of strongly hydrophobic therapeutical agents, which is attributed to their ability to form large mixed micelles that can trap the bioactive agents inside (McClements, 2021). It is also important to carefully selected the type of emulsifier used to ensure that small droplets can be formed during homogenization, the systems remain stable during storage, and the droplets do not undergo extensive aggregation within the gastrointestinal tract (as this can reduce bioavailability by restricting the access of digestive enzymes to the lipids). It may also be important to include other additives, such as antioxidants, to preserve the therapeutic agents during storage. Emulsions and nanoemulsions are typically produced using mechanical devices known as homogenizers, such as high shear mixers, colloid mills, high pressure valve homogenizers, sonicators, and microfluidizers (McClements, 2011; McClements & Rao, 2011). Emulsions and nanoemulsions are thermodynamically unstable and may be broken down through a variety of physical and chemical instability mechanisms, including creaming, sedimentation, flocculation, coalescence, and Ostwald ripening (McClements, 2011; McClements & Rao, 2011). Consequently, they must be carefully formulated to avoid these problems. After formation, emulsion-based systems are typically in a fluid form. They can be converted into gels by adding gelling agents or promoting aggregation of the oil droplets. They can be converted into powders through dehydration, which is usually carried out commercially using spray drying technologies. The functional performance of emulsions and nanoemulsions can be improved by using structural design methods to generate more sophisticated morphologies, such as multilayer emulsions, multiple emulsions, Pickering emulsions, or filled microgels (Tan & McClements, 2021). However, these advanced emulsion technologies are more costly to prepare and therefore they should only be utilized when required.
Nanostructured lipid carriers (NLCs) and solid lipid nanoparticles (SLNs) are oil-in-water emulsions or nanoemulsions where the lipid has been partially or fully solidified, respectively (McClements & Li, 2010). They are typically prepared using the same homogenization methods as used to produce emulsions and nanoemulsions (Weiss, Decker, McClements, Kristbergsson, Helgason & Awad, 2008). However, the lipid phase is comprised of a high melting point lipid. Typically, an emulsion or nanoemulsion is first formed at a high temperature (above the melting point of the lipid), then it is rapidly cooled to form NLCs or SLNs. However, the lipid phase must be carefully selected to ensure that the lipophilic therapeutic agent is not expelled and the lipid droplets do not aggregate after the lipid phase is solidified (Weiss, Decker, McClements, Kristbergsson, Helgason & Awad, 2008). Well-designed SLNs and NLCs are often more effective at preserving encapsulated lipophilic compounds against chemical degradation during storage, which is attributed to the ability of the solidified lipid phase to inhibit molecular diffusion (Weiss, Decker, McClements, Kristbergsson, Helgason & Awad, 2008). Encapsulation of melatonin in SLNs has also been shown to lead to a more sustained release profile after oral ingestion (Priano et al., 2007), to increase its bioavailability after oral administration to humans (Mistraletti et al., 2019), and to increase its antioxidant activity after dermal application (Mirhoseini et al., 2019). Stearic acid-based SLNs have been used to encapsulate resveratrol and increase its bioavailability after oral administration to Wistar rats (Pandita, Kumar, Poonia & Lather, 2014). Furthermore, NLCs have been shown to increase the bioactivity of melatonin in in vitro  fertilization media (Siahdasht, Farhadian, Karimi & Hafizi, 2020).
Microemulsions contain small spheroidal particles consisting of a hydrophobic core and a hydrophilic shell, which are primarily made up from surfactants (McClements, 2020a). The non-polar tails of the surfactants cluster together through hydrophobic attraction and form a hydrophobic core, while the polar heads of the surfactants form a hydrophilic shell that ensures their water-dispersibility. Lipophilic therapeutic agents, like resveratrol or melatonin, can be incorporated into the hydrophobic core of microemulsions (Nemen & Lemos-Senna, 2011). These colloidal systems are thermodynamically stable and can often be formed by simply mixing the ingredients together, once the optimum composition has been established. They tend to be optically clear because the size of the particles (< 50 nm) is typically much less than the wavelength of light. Encapsulation of resveratrol in microemulsions has been shown to increase its water-dispersibility, photostability, and antioxidant activity (Juskaite, Ramanauskiene & Briedis, 2017; Lv et al., 2018). The encapsulation of melatonin in microemulsions has been shown to increase its bioavailability after being applied to the skin of human patients (Mistraletti et al., 2019). The main drawback of microemulsions is that they usually have to be formed from relatively high concentrations of non-ionic surfactants, which can cause taste, cost, or toxicity problems.
Liposomes (d > 200 nm) and nanoliposomes (d < 200 nm) are spheroidal particles that typically have an onion-like (vesicular) structure, which are formulated from phospholipids (McClements, 2020a). These liposomal systems are comprised of one or more concentric phospholipid bilayers surrounding an aqueous core. The bilayer structures tend to form spontaneously when the phospholipids are mixed with water due to the hydrophobic effects. However, some form of processing is need to create a dispersion of relatively small and uniform liposomes or nanoliposomes, such as microfluidization. Liposomal systems can encapsulate hydrophilic and hydrophobic therapeutic agents because they have both polar and non-polar regions inside them. Hydrophobic molecules like melatonin and resveratrol are usually located within the non-polar regions formed by the tails of the phospholipids in the bilayer membranes. Encapsulating resveratrol within a liposomal formulation has been shown to prolong its release and increase its therapeutic effects in rat models of nerve injury (Feng, He, Mao, Shui & Cai, 2019). Metformin-encapsulated liposomes enhance therapeutic efficacy and clinical use of metformin against breast cancer cells (Khiavi, Safary, Barar, Ajoolabady, Somi & Omidi, 2020; Shukla et al., 2019).
Cyclic oligosaccharides, known as cyclodextrins, consist of glucopyranose units with α (1→4) bonds (Astray, Gonzalez-Barreiro, Mejuto, Rial-Otero & Simal-Gandara, 2009). In aqueous solutions, cyclodextrins have a hydrophobic cavity than can incorporate non-polar therapeutic agents and a hydrophilic exterior that ensures their water-dispersibility. Inclusion complexes formed between α-/β-/γ-cyclodextrins and resveratrol have been shown to significantly improve the water-dispersibility of resveratrol (Bertacche, Lorenzi, Nava, Pini & Sinico, 2006; Silva et al., 2021). Researchers have also shown that cyclodextrins can also be used to increase the water-dispersibility of melatonin (Grygorova et al., 2019). Studies have confirmed that cyclodextrins and resveratrol form 1:1 complexes with the therapeutic molecule trapped in the hydrophobic cavity (Lucas-Abellán, Fortea, Gabaldón & Núñez-Delicado, 2008). An in vivo study showed that encapsulation of resveratrol in cyclodextrins increased its anticancer activity against cervical cancer (Hao et al., 2021), which was probably because the delivery system increased the amount of the therapeutic agent reaching the site of action in an active form.