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.