marine drugs

Review
Microalgae Encapsulation Systems for Food,
Pharmaceutical and Cosmetics Applications
Marta V. Vieira , Lorenzo M. Pastrana and Pablo Fuciños *
Food Processing and Nutrition Group, International Iberian Nanotechnology Laboratory, Av. Mestre José Veiga s/n,
4715-330 Braga, Portugal; marta.vieira@inl.int (M.V.V.); lorenzo.pastrana@inl.int (L.M.P.)
* Correspondence: pablo.fucinos@inl.int




Received: 15 November 2020; Accepted: 8 December 2020; Published: 15 December 2020


Abstract: Microalgae are microorganisms with a singular biochemical composition, including several
biologically active compounds with proven pharmacological activities, such as anticancer,
antioxidant and anti-inflammatory activities, among others. These properties make microalgae
an interesting natural resource to be used as a functional ingredient, as well as in the prevention and
treatment of diseases, or cosmetic formulations. Nevertheless, natural bioactives often possess inherent
chemical instability and/or poor solubility, which are usually associated with low bioavailability.
As such, their industrial potential as a health-promoting substance might be severely compromised.
In this context, encapsulation systems are considered as a promising and emerging strategy to overcome
these shortcomings due to the presence of a surrounding protective layer. Diverse systems have already
been reported in the literature for natural bioactives, where some of them have been successfully
applied to microalgae compounds. Therefore, this review focuses on exploring encapsulation systems
for microalgae biomass, their extracts, or purified bioactives for food, pharmaceutical, and cosmetic
purposes. Moreover, this work also covers the most common encapsulation techniques and types of
coating materials used, along with the main findings regarding the beneficial effects of these systems.

Keywords: bioactive; drug delivery systems; functional food; cosmeceuticals

1. Introduction
Microalgae are a heterogeneous group of photosynthetic microorganisms, whose evolutionary
and phylogenetic diversity has provided a vast assortment of biochemical compositions [1].
These microorganisms are able to biosynthesize, accumulate and secrete a great range of primary and
secondary metabolites as a response to changes in the external environment, many of which are highly
valuable substances with industrial applications and health benefits [2].
The use of microalgae by humans dates back thousands of years, where it was used as a food
source by different populations; nevertheless, the commercial exploitation of this resource is only a few
decades old, when there was an apprehension regarding a possible insufficient protein supply due to
the rapid increase in the world population [3,4]. Microalgae are well-known for their high protein and
nutritional content, but more recently, studies have been focused on the unique biologically active
compounds produced by their species, such as polyunsaturated fatty acids, pigments, antioxidants,
polyphenols, polysaccharides, and other equally important substances [3,4].
Lately, there has been a growing trend towards using natural ingredients in food,
pharmaceutical, and cosmetic industries due to the increasing concerns regarding consumer safety,
environmental sustainability, and regulatory issues over the introduction of synthetic chemicals
in human nutrition, healthcare, and beauty products [5–7]. Several microalgae bioactives possess
significant biological activities, including anticancer, antioxidant, anti-inflammatory, antimicrobial,
and immunomodulatory activities, among others [8–10]. Therefore, the use of such compounds

Mar. Drugs 2020, 18, 644; doi:10.3390/md18120644 www.mdpi.com/journal/marinedrugs

Mar. Drugs 2020, 18, 644 2 of 44

seems to be a promising and innovative approach to the development of healthier, functional,

and sustainable products.
Overall, microalgae may be proposed to obtain commodities with existing market value,
refined bioactives, or the whole cell could even be the target product [11]. Yet, purified compounds or
bioactive extracts are usually chemically unstable and strongly susceptible to oxidative degradation,
particularly when exposed to oxygen, light, moisture, extreme pH, and high temperatures. The oxidative
degradation may also deteriorate the compounds, leading to the development of unpleasant tastes
and off-odours in the fortified product and, subsequently, may result in a negative effect on shelf
stability, sensory characteristics, and consumer acceptability of the product [12]. Moreover, the low
bioavailability and poor water solubility are usually recurrent issues related to the application of
microalgae bioactives; in pharmaceutical and functional food products, for instance, the absorption
of these compounds may be hindered due to gastrointestinal tract conditions, as well as due to their
physicochemical properties [13,14].
These developmental and technological issues address the importance of researching strategies
to preserve the functionality of microalgae bioactives from processing until they reach their target
site. In this context, encapsulation systems are considered a promising approach, which have
been applied successfully in diverse fields. The process of encapsulating a bioactive consists of its
entrapment within one or more coating materials through different techniques, resulting in nano-
or microparticles [15]. This strategy is associated with several advantages, including protecting
the bioactive compound during processing, storage, and distribution; promoting release control;
masking off-flavours; improving solubility and bioavailability, among others [16,17].
Considering the above mentioned, the present review aimed at describing the encapsulation
systems reported in the literature of different microalgae biomass, its extracts, or purified compounds,
focusing on food, pharmaceutical, and cosmetic applications. The described techniques of encapsulation,
types of coating material, and the main findings regarding the beneficial effects of these systems were
also considered.

2. Microalgae
Microalgae are single-celled, ubiquitous, prokaryotic, and eukaryotic primary photosynthetic
microorganisms, which are taxonomically and phylogenetically diverse [9,18]. They are ancestral
living organisms that have adapted uniquely to extreme habitats over billions of years of evolution
and can be found almost anywhere on Earth; in freshwater, seawater, and hypersaline environments,
but also in moist soils and rocks [19]. Their classification is based on various properties,
such as pigmentation, the chemical nature of photosynthetic storage products, the organization of
photosynthetic membranes, and other morphological features. The most abundant microalgal classes
are Cyanophyceae (blue-green algae), Chlorophyceae (green algae), Bacillariophyceae (including the
diatoms), and Chrysophyceae (including golden algae) [3,20]. A resume of the main microalgae classes,
their most studied species, and associated biological activities are described in Figure 1.
Interest in microalgae cultivation has been prospering globally in recent decades for diverse reasons.
There are several industrial and commercial applications associated with these microorganisms and
examples of success include formulations in different sectors, such as functional foods, feed, cosmetics,
pharmaceuticals, and fertilizers; as well as tools for wastewater treatment and biofuel production [21,22].
Moreover, many advantages have already been reported involving their cultivation process in
comparison with other feedstocks. Firstly, microalgae reproduce themselves using photosynthesis to
convert sun energy into chemical energy, completing an entire growth cycle every few days. Secondly,
they can grow almost anywhere, requiring mostly sunlight and some simple nutrients; although the
process can be accelerated heterotrophically by the addition of specific nutrients and changes in
cultivation parameters. Accordingly, microalgae have much higher growth rates and productivity
when compared to conventional forestry, crops, and other aquatic plants, demanding much less
land area [23,24].
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Figure 1. Main microalgae classes, their most important species, and associated biological activity
(microalgae images were obtained from the “Microalgae strain catalogue”, second edition, published in
the Enhance Microalgae Project, available at https://www.enhancemicroalgae.eu/wp-content/uploads/
2020/05/EMA-Strain-catalogue-2nd-Edition.pdf).

Through adaptive evolution and metabolic diversity, microalgae have developed a wide range
of high value biologically active compounds, comprising pigments, antioxidants, polysaccharides,
triglycerides, fatty acids, and vitamins [25]. It is estimated there are 70,000 to one million microalgae
species; however, only about 44,000 have already been described. Furthermore, from those, only a
limited number have been studied for commercial purposes [26]. Some of the most biotechnologically
relevant microalgae are the green algae (Chlorophyceae) Chlorella vulgaris, Haematococcus pluvialis,
Dunaliella salina, and the Cyanobacteria Arthrospira platensis, which are broadly commercialized,
mainly as nutritional supplements for humans and as animal feed additives [27].
The multicellular filamentous Cyanobacteria from the genus Arthrospira (formerly known as
“Spirulina”) occur naturally in alkaline lakes and ponds, being widely cultured around the world.
The two most important species of Arthrospira, A. maxima, and A. platensis, are commonly applied both
as a functional ingredient in food preparations and as a source of the blue photosynthetic pigment
C-phycocyanin, which is used in cosmetics and the food industry [28]. This species has been used as a
nutrient-rich (especially vitamin B12 and proteins) food source with the oldest records indicating use
by the Aztecs, who harvested this microalga from Lake Texcoco in Mexico; and by the local people in
Lake Chad, who used A. platensis as a nutritional supplement known as “dihe” [6,29]. Apart from
its significance as a food additive, A. platensis is also recognized by the broad range of potential
medical and pharmaceutical applications attributed to its metabolites. Studies have evidenced several
biological activities, such as antitumor, antibacterial, anti-inflammatory and hepatoprotective activities,
among others, directly related to the antioxidant capacity ascribed for C-phycocyanin and other
compounds [30,31]. Similarly, the freshwater unicellular blue-green microalga Aphanizomenon flos-aquae,
which grows spontaneously in Upper Klamath Lake in Oregon, USA, is also consumed as a nutrient-rich
food source and for its health properties. Similar to the Arthrospira species, A. flos-aquae is an important
source of the pigment C-phycocyanin; hence, demonstrating a strong antioxidant potential [32,33].
Mar. Drugs 2020, 18, 644 4 of 44

The unicellular green alga Chlorella is one of the largely studied microalgae due to their
biotechnological importance as a valuable source of nutrients. Species from this genus were one of the
first microalgae considered for mass cultivation and the first microalga produced commercially.
Chlorella cells actively growing under normal conditions are typically rich in protein (40–60%)
and are largely made up of essential amino acids, with a profile that suits human nutrition. In this
context, Chlorella biomass may be considered as a desirable candidate for protein supplements or
single-cell protein [34,35]. Furthermore, these species are also rich in carotenoids, vitamins and
other bioactives, demonstrating potential health benefits, such as efficacy on gastric ulcers, wounds,
and constipation; preventive action against both atherosclerosis and hyper-cholesterol; and antitumor
activity. The suggested most important active compound is β-1,3-glucan, which is believed to be an
active immune-stimulator, free radical scavenger, and a reducer of blood lipids [4,36,37].
Another important member of the green algae class is the freshwater unicellular microalga
Haematococcus pluvialis. Under extreme environmental conditions, such as high-intensity light
or oligotrophic circumstances, this species undergoes several morphological and biochemical
modifications, including an intense biosynthesis of the carotenoid astaxanthin [38]. In the last
few decades, H. pluvialis has received significant attention from the scientific and biotechnological
communities for being considered as the most significant biological source of that carotenoid in
nature [39,40]. Astaxanthin is a natural pigment with several applications in the nutraceutical, cosmetic,
food, and feed industries [41]. Moreover, it also possesses a powerful antioxidant potential due to its
unique chemical configuration, which is associated with assorted biological activities demonstrated in
both animal and clinical studies [42]. Many authors have already described astaxanthin’s valuable
effects in inflammatory responses and the immune system, in hypertension, cancer, ocular and
cardiovascular diseases, as well as in skin ageing defence [43–45].
Regarding carotenoid production, the unicellular green microalga Dunaliella salina is equally
important for its recognition as the richest source of natural β-carotene [46]. When exposed to specific
extreme environmental conditions, such as high-intensity light, high salinity, extreme temperatures,
and/or nutrient deprivation, D. salina can accumulate an exceptionally large amount of β-carotene
(up to 14% of the dry algal biomass), resulting in orange-coloured cells. This great carotene productivity
has led to the large-scale application of D. salina for commercial production of natural β-carotene,
widely used as an antioxidant and colourant in the food, feed, cosmetics, and pharmaceutical
industries [47,48]. Additionally, this species also contains other important lipid components, glycerol,
proteins, and carbohydrates [49].
In the context of microalgae importance in different fields, some species have not been
fully explored, but have been demonstrated to be a promising source of bioactives according to
published studies. The microalga Phaeodactylum tricornutum is a marine diatom, which accumulates
eicosapentaenoic acid (EPA, 20:5n-3) as a major component of its fatty acid content [50]. This species
is also a rich source of the carotenoid fucoxanthin, whose intake has been suggested to
improve insulin resistance and to decrease the blood glucose level, along with anticancer and
anti-inflammatory effects [51,52]. Furthermore, some other microalgae genera, such as Nannochloropsis,
Tetraselmis, Scenedesmus, and Isochrysis, have revealed their importance due to the production of
long-chain fatty acids, i.e., docosahexaenoic acid (DHA) and EPA, representing also a source of
antioxidant compounds [53,54].
Although there are a very large number of red algae (Rhodophyta) in nature, only a few species
represent the microalgae group. The genus Porphyridium is the most studied one due to the particular
interest in its species as a source of sulphated polysaccharides, proteins, the polyunsaturated fatty
acids (PUFAs) arachidonic acid and EPA, and the phycobiliprotein phycoerythrin [35]. Studies have
demonstrated that the sulphated polysaccharides of Porphyridium sp. exhibit potential antiviral activity
against herpes simplex virus (HSV-1 and 2) both in vitro and in vivo [55,56]. Furthermore, it has also
been reported that different-molecular-weight subunits of its polysaccharides demonstrate important
antioxidant and immunomodulatory activities [57,58]. Likewise, the Cyanobacteria Phormidium sp.
Mar. Drugs 2020, 18, 644 5 of 44

is a recognized source of extracellular polymeric substances (EPS), which possess applications in the
pharmaceutical, cosmetic, and food industries as an emulsifier and thickening agent [59]. Additionally,
species of this genus have been reported to inhibit the growth of different Gram-positive and
Gram-negative bacterial strains, yeasts, and fungi [60].

Biochemical Composition
Microalgae produce a suite of biochemical molecules, and the cellular content of each fraction
varies according to the specific strain of alga and their physiological responses to biotic and abiotic
factors, e.g., light intensity, photoperiod, temperature, nutrients, and growth phase [61,62]. In fact,
these factors not only affect photosynthesis and cell biomass productivity, but also influence the pattern,
pathway, and activity of the cellular metabolism, which, consequently, modify the cell composition [63].
As such, due to their evolutionary and phylogenetic diversity, combined with the possibility of
manipulating cultivation parameters to stimulate compounds’ biosynthesis, these microorganisms
became extremely attractive for bioprospecting and potential exploitation as commercial sources of a
wide range of biomolecules [1,64].
Bioactive compounds of microalgal origin can be sourced directly from primary metabolisms,
such as proteins, fatty acids, and vitamins; or can be synthesized from secondary metabolism.
Such compounds can present several biological activities, which might be used in the reduction and
prevention of diseases (Figure 1). In most microalgae, bioactive compounds are accumulated in the
biomass; however, in some cases, these metabolites are excreted into the medium, being known as
exometabolites [2].
Chemically, microalgae compounds can be grouped into proteins/enzymes, lipids/fatty acids,
carbohydrates, pigments, vitamins, minerals, and other compounds not included in these classes [65].
Even though the biochemical differences in microalgal classes and species are evident, protein is typically
the major organic constituent (12–35%), normally followed by lipids (7–23%) and carbohydrates
(5–23%). Yet, these proportions can drastically change under specific environmental conditions,
as previously mentioned [63].
Proteins play an important role in the structure and metabolism of microalgal cells. They are
a fundamental component of the membrane and light-harvesting complex, including numerous
catalytic enzymes involved in photosynthesis [57]. The protein content of many species can
compete, quantitatively and qualitatively, with conventional protein sources. In terms of quantity,
several microalgae are reported to possess very high concentrations of protein, ranging from 42% to
over 70% in certain Cyanobacteria, and up to 58% in Chlorella vulgaris on a dry weight basis. In terms of
quality, microalgae contain all of the essential amino acids that mammals are unable to synthesize [61,66].
Moreover, some proteins, peptides, and amino acids also have biological functions associated with
nutritional benefits and human health. Thus, these biopolymers can be used as nutraceuticals or included
in functional food formulations [65].
Among the biochemical components, lipids have received the greatest attention regarding
extraction and commercialization. When research on algal lipids first began, the major goal was
aimed at biodiesel production. Nevertheless, the significant amount of polyunsaturated fatty acids
(PUFAs) present in microalgae composition have provided considerably more commercial value to
these bioactives as a nutraceutical and infant formulation supplement [61].
Microalgae lipid fraction is mainly composed of neutral and polar lipids, whose proportion varies
along with the different growth phases, species, and environmental/culture conditions. Polar lipids
possess a structural function, comprising the cell wall and organelle membranes, such as glycolipids
and phospholipids [67]. On the other hand, neutral lipids are regarded as energy storage products,
which include acylglycerols (mono-, di- and triglycerides), sterols, hydrocarbons, free fatty acids,
and pigments [61,68]. The fatty acids in microalgae are biosynthesized through the addition of acetate
(C-2) units; almost all are straight-chain and with an even number of carbon atoms, predominantly
between C-12 and C-22. The main saturated fatty acids present in these structures are acids with 12, 14,
Mar. Drugs 2020, 18, 644 6 of 44

16, and 18 carbon atoms. A wide variety of unsaturated fatty acids are found in algae, with chains
between 16 and 22 carbon atoms and double bonds in cis configuration [64].
Microalgae produce an interesting array of fatty acids, and they are reported to be the primary
producers of some PUFAs in the biosphere, mainly omega (ω)-3 long-chain polyunsaturated fatty
acids [69]. The importance of these compounds is based on the inability of humans to synthesize
part of them; PUFAs play a key role in several bodily functions and processes, acting as a precursor
of distinct biological molecules [70]. Examples of PUFAs produced by microalgae include the
linolenic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) ω-3 fatty acids; and the linoleic,
gamma-linolenic (GLA) and arachidonic (ARA) ω-6 fatty acids [71].
Under optimal cultivation conditions, several species, especially those belonging to the genera
Botryococcus, Chlorella, Nannochloropsis, Neochloris, Nitzschia, Scenedesmus, Isochrysis, Dunaliella and
Schizochytrium, are described to show exceptionally high amounts of lipids in their cell mass.
Regarding industrial applications, the eukaryotic microalgae Chlorella vulgaris (up to 58% dry weight),
Nannachloropsis oculata (up to 69% dw), Botryococcus braunii (up to 75% dw), and Scenedesmus obliquus
(up to 50% dw) have been reported to be promising lipid sources [72,73].
The colourful appearance of microalgae is derived from the presence of pigments, which absorb
visible light and have a fundamental role in cell photosynthetic metabolism. The three major classes of
these compounds are chlorophylls, carotenoids, and phycobiliproteins [74,75]. Chlorophyll -a is the
primary pigment in all photosynthetic organisms; it absorbs most energy from the wavelengths of
violet-blue and orange-red light, serving as a primary electron donor in the electron transport chain [76].
All microalgae contain one or more types of chlorophyll, which are classified according to their
structural features and wavelength absorption [77]. The type -a is the only one found in Cyanobacteria
and Rhodophyta, and the types -a and -b are found in Chlorophyta and Euglenophyta. Chlorophylls -c,
-d and -e can be found in diverse marine microalgae and freshwater diatoms. The chlorophyll fraction
usually represents about 0.5–1.5% of the cell dry weight [64].
Carotenoids are fat-soluble substances with colours varying from brown, red, orange, to yellow.
These pigments perform two key roles in photosynthesis: the light absorption in regions of the visible
spectrum and the photoprotection of the photosynthetic systems. All carotenoids directly involved in
photosynthesis are called primary carotenoids, where they participate in the transferring of absorbed
energy to chlorophylls; thus, expanding the light-absorbing spectrum of the cell. Primary carotenoids
are structural and functional components of the cellular photosynthetic apparatus, making them
essential for the survival of the cells [75].
Some microalgae species can also undergo a carotenogenesis process as a response to
different environmental factors and culture stresses, e.g., high-intensity light, nutrient deprivation,
and temperature changes [78]. These substances are categorized as secondary carotenoids, and they
play a major role in cell protective mechanisms through the dissipation of most energetic states of
chlorophyll, occasioned by excessive absorption of light [79,80]. The presence of these carotenoids
hinders the formation of reactive oxygen species (ROS), providing these pigments with a significant
antioxidant property. Examples of primary carotenoids are α-carotene, β-carotene, lutein, violaxanthin,
zeaxanthin, and neoxanthin, whereas typical secondary carotenoids include astaxanthin, canthaxanthin,
and echinenone [77,81].
In addition to chlorophyll and carotenoid, the pigment-protein complex phycobiliprotein is
also commonly present in Cyanobacteria, Rhodophyta, and Cryptomonads. These complexes
are deep-coloured water-soluble fluorescent cell components, which belong to the photosynthetic
light-harvesting antenna [82]. According to their amino acid sequences and absorption spectrum,
phycobiliproteins can be divided into four main classes, namely allophycocyanin (bluish-green),
phycocyanin (blue), phycoerythrin (red), and phycoerythrocyanin (orange) [83,84]. The principal
producers of microalgal pigments are the species A. platensis, P. cruentum, H. pluvialis, and D. salina,
which are able to accumulate a significant amount of phycocyanin, phycoerythrin, astaxanthin,
and β-carotene, respectively.
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Equally representing a great fraction of the microalga cell, carbohydrates are the major products
derived from photosynthesis and carbon fixation metabolism. These constituents are either accumulated
in the plastids as reserve materials, e.g., starch; or become the main component of cell walls, such as
cellulose, pectin, and sulphated polysaccharides [85,86]. A third possibility is the excretion of
large amounts of polysaccharides into the extracellular medium, known as exopolysaccharides
(EPS), supposedly in order to protect the cell from fluctuations in environmental conditions
and/or predators [87].
The biomass carbohydrate content, similar to other microalgal compounds, also depends on
the species and on the cultivation and environmental conditions. Green microalgae, for instance,
synthesize amylopectin-like polysaccharides (starch) as reserve carbohydrates; Cyanobacteria synthesize
glycogen (α-1,4 linked glucan), and red microalgae produce a polymer known as floridean starch (a hybrid
of starch and glycogen) [88,89]. Moreover, a commonly found polysaccharide in a large number of species
is chrysolaminarin, a linear polymer of β(1→3), and β(1→6) linked glucose units [89].
In addition to bio-macromolecules, microalgae constitute a valuable source of vitamins and
minerals. Vitamin A, B1 , B2 , B6 , B12 , C, E, K, niacin, nicotinate, biotin, and folic acid are some of the
examples found in these micro-organisms. In some microalgae genus, such as Arthrospira, Chlorella,
and Scenedesmus, vitamin A, B1 , B2 , E, and niacin can achieve even higher levels than those found in
vegetables [85]. Concerning the minerals’ content, it may represent around 2.2 to 4.8% of the total
microalgae biomass dry weight, including calcium, phosphorous, magnesium, potassium, sodium,
zinc, iron, copper, and sulphur [57]. Furthermore, several microalgae exhibit a high content of different
phytochemicals compounds, such as polyphenols, alkaloids, among others [90,91].

3. Encapsulation
Encapsulation may be defined as a process in which a substance (active agent) is entrapped or
coated by a carrier material, in order to form a particulate system. The encapsulated compounds are
also designated as the core, fill, or internal phase, whereas the carrier substances can be identified as
wall material, membrane, capsule, shell, matrix, or external phase [12]. This technique may be used to
encapsulate compounds in the solid, liquid, or gaseous state in small particles, which can be classified
as nanoparticles when the dimensions vary from 1 to 100 nm; or microparticles, when the dimensions
range from 100 nm to 1000 µm [92,93].
The protective barrier provided by encapsulation offers several advantages. The primary reason
to develop these particulate systems is to maintain the biological, functional, and physicochemical
properties of the active agent [94]. The wall material serves as a protection from adverse environmental
and processing conditions, such as the undesirable effect of light, temperature, moisture, and oxygen;
therefore, contributing to an increase in stability and an extended shelf-life [95]. Another important
advantage is the possibility of overcoming challenges that normally restrict the incorporation of certain
substances into commercial products. Encapsulation allows the increase in solubility of a compound
into a dissimilar medium, masking unpleasant flavours, enhancing the bioavailability and bioactivity,
as well as controlling and targeting the release of the core material as a response to external conditions
(pH, temperature, etc.) [15,94,96].
The retention of the core substance within a particle and its stability depends on several factors,
comprising the desired physicochemical and functional properties of the final encapsulation system,
along with the properties of the carrier material and the active agent. The major characteristics to
be considered regarding the core and coating materials are their chemical nature, molecular weight,
polarity, and solubility; while for the final particulate system, the entrapment efficiency, permeability,
degradability, and release profile must be equally taken into account [97–99].
Encapsulation technology has been extensively researched and applied in diverse areas, such as
the pharmaceutical, medical, food, cosmetics, chemical, and agricultural industries [98,100]. In the
pharmaceutical field, for instance, encapsulation is a key strategy to assist specific drawbacks in the
formulation development, as it is capable of promoting drug delivery to specific body sites, control drug
Mar. Drugs 2020, 18, 644 8 of 44

release, act as diagnostic tools, and improve physicochemical properties, e.g., water solubility, which,
in turn, can bring positive changes to the medical treatment, such as lowering the therapeutic dose
and minimizing the side effects [101–104]. Similar advantages can be related to the application of
encapsulation in the cosmetics segment, where particulate systems could lead to a sustained release of
the active agent, as well as an enhanced and deeper skin penetration [105,106].
Likewise, the food industry can particularly benefit from the use of encapsulation,
especially regarding the development of functional foods. The addition of biologically active
compounds into food has emerged as an exciting health-promoting strategy in recent decades;
however, it may present several limiting factors, including high sensitivity to processing conditions,
short shelf-life, fast-release of flavour during storage, limited uptake and bioavailability, lack of
compatibility and uniformity with the food matrix, or degradability through the gastrointestinal tract
passage [16,107,108]. In light of this, encapsulation represents a useful tool for the suppression of the
aforementioned limitations, since it enables the protection of a wide range of compounds by their
entrapment into a protective matrix [109].

3.1. Structure and Composition

The structure of an encapsulation system depends upon the arrangement of the core substance
and deposition process of the coating material, which can be broadly divided into reservoir or matrix
systems [16]. The reservoir systems are also referred to as nano- or microcapsules and can be further
classified into mono-core, multi-core, or multi-shell mono core; where the first contains an outer shell
around a single core, the second has distinct cores entrapped into a shell, and the last comprises a
single core surrounded by many shells. On the other hand, the matrix system is when the active agent
is uniformly distributed within the coating material network. The particulate systems are normally
formed as spherical shapes; however, non-spherical/irregular configurations can be found for all the
systems previously described [15,110]. A resume of all these structures is demonstrated in Figure 2.

Figure 2. Different encapsulation systems structures: (a) mono-core, (b) multi-shell mono-core,
(c) multi-core, (d) matrix and (e) representative of an irregular shape particle. Adapted from [111].

Regarding the composition of an encapsulation system, the encapsulated active agents can
have a hydrophilic or lipophilic nature, comprising several classes, such as drugs, vitamins,
minerals, nutraceuticals, antimicrobials, antioxidants, flavours, enzymes, essential oils, colourants,
among others [93]. Additionally, the coating material can be selected from a wide variety of natural
and synthetic polymers, co-polymers, and bio-based substances. This choice will depend on the active
agent to be encapsulated and the properties desired for the final system [112]. Important aspects to
be considered are mostly the solubility, stability, release properties, and safety; thus, for use within
the food industry, the substance must have a food-grade status; and for pharmaceutical applications,
it should present, among other criteria, biocompatibility and biodegradability [113]. A resume of the
most used coating materials for encapsulation purposes can be found in Table S1.
Materials derived from natural sources may be classified as (i) carbohydrates, such as starch,
maltodextrin, pectin, cellulose, cyclodextrin, and inulin; (ii) proteins, such as gelatine, whey protein,
casein, bovine serum albumin, and different vegetable sources; (iii) waxes or fats, including glycerides
and phospholipids, or (iv) gums, such as Arabic, guar, and mesquite. In addition to these,
Mar. Drugs 2020, 18, 644 9 of 44

the polysaccharides chitosan and alginate are mostly investigated for food and pharmaceutical
applications [16,112,114]. The main advantages of using these bio-based materials are that they are
normally abundant in nature, biodegradable, and biocompatible. On the other hand, as they represent
natural resources, they can display unstable properties due to batch to batch variation [15].
Another range of coating materials used for the encapsulation process is synthetic polymers,
which can be further classified into biodegradable and non-biodegradable. Among the biodegradables
ones, aliphatic polyesters, such as poly (ε-caprolactone) (PCL), poly (lactic acid) (PLA), and poly
(lactic-co-glycolic acid) (PLGA) are frequently explored in the pharmaceutical industry [115].
Other examples of this group also comprise the polyanhydrides, polyamides, polyurethanes,
and phosphorus-based polymers. Concerning the non-biodegradable polymers, cellulose derivatives,
such as carboxymethyl cellulose, ethyl cellulose, or cellulose acetate, are also broadly applied, along with
poly(ethylene glycol) (PEG), polyvinyl alcohol (PVA), and poly(N-vinylpyrrolidone) (PVP) [113,116].
Differing from natural materials, synthetic polymers exhibit a higher chemical and mechanical stability,
with the possibility to modify their properties according to the desired final system. Nevertheless,
low biocompatibility and biodegradability may represent the main drawbacks [15].
Furthermore, wall materials can also suffer a functionalization process, which offers the possibility
to obtain encapsulation systems with modified properties, different from those normally found in the
literature, e.g., an increase in biodistribution and additional bio-marker function [117,118].

3.2. Encapsulation Techniques

Several techniques have been proposed in the literature for encapsulation processes; nevertheless,
there is not a specific method that could be regarded as a standard and suitable for the
different active agents and encapsulated systems. Before choosing a technique, the crucial
aspects to be analysed are the type and properties of the core and coating materials, and the
proposed application and characteristics of the final system [12,97,119]. Moreover, the size,
shape, and internal structure of the particles vary considerably depending on the selected
production method [120]. Some of the most described encapsulation techniques are spray-drying,
spray-cooling/chilling, fluidized bed, coacervation, solvent evaporation, liposomes, supercritical fluid
technology, interfacial polymerization, nanoprecipitation, emulsification, inclusion complexation,
electrospray system, and extrusion [94–96,117,119]. It is noteworthy mentioning that for a widespread
application, the chosen method should be cost-effective, easy to scale-up, and have its safety
status considered [99].
The strategies involved in the synthesis and production of encapsulation systems are based
on two main approaches, namely bottom-up and top-down. The bottom-up approach is defined
when large structures are built or grown through atom-by-atom or molecule-by-molecule techniques,
mediated by different interactions, e.g., van der Waals, ionic and hydrophobic interactions [121].
This approach includes chemical synthesis, self-assembly, and positional assembly of molecules,
which are influenced by several physicochemical and environmental factors, such as pH, temperature,
concentration, and ionic strength [121,122]. Examples of techniques that follow this approach are
nanoprecipitation, coacervation, inclusion complexion, and supercritical fluid encapsulation [123].
Conversely, the top-down approach involves physical processing of the core and coating materials,
which requires precise tools focusing on the size reduction and shaping of the structure for the
desired application. Extrusion, homogenization, electrospinning/spraying, and emulsification-solvent
evaporation are examples of top-down techniques [120,122,123]. In general, the bottom-up techniques
are considered more advantageous than the top-down approach, as they allow greater control over
the properties of the particles, namely the size, morphology, and physical state, and they are also less
energy-consuming. Moreover, the risk of sample contamination is often significantly reduced compared
to top-down technologies [99,124]. A schematic representation of the main reported encapsulation
techniques of both approaches is shown in Figure 3.
Mar. Drugs 2020, 18, 644 10 of 44

Figure 3. Encapsulation techniques of top-down and bottom-up approaches. Adapted from [99].

Spray drying is one of the oldest and the most widely used encapsulation techniques, mainly in the
food sector. Firstly, the active agent is dispersed or dissolved in an aqueous solution or one prepared
with the coating material. The mixture is then atomized, where little droplets are formed and dried
through hot circulating air [109]. The size of the particles normally varies from 1 to 50 µm, but it can be
reduced to 0.2 µm by using a nano spray dryer. The encapsulation efficiency is influenced by different
process parameters, including the viscosity and surface tension of the solution, the solubility of the
core, or even the air entrance temperature, air flux, and humidity [125]. The main advantages of this
technique are its simplicity, flexibility, fast production, and low operating costs. On the other hand,
the particles formed may not be uniform and some sensitive compounds could be degraded by the
high air temperatures [92].
The extrusion technique is another quite common choice to obtain an encapsulation system.
It consists in the passage of a solution composed of the coating material and active agent through
a nozzle, reaching a gelling environment. Several methods have been used to form extruded
particles, including electrostatic extrusion, simple dripping, vibrating jet/nozzle, and melt extrusion.
Following the particle formation, they must be instantly hardened to capsules by either physical
processes, e.g., cooling or heating, or chemical processes, e.g., gelation [110]. Extrusion is a simple
Mar. Drugs 2020, 18, 644 11 of 44

and low-cost technique, which is suitable for labile substances when only a final gelation process is
required, yet it may present low encapsulation efficiency [126].
Coacervation consists of the formation of two immiscible phases from a solution containing a
dispersed polymer. The substance to be encapsulated is dispersed in a polymeric solution, which will
act as the coating material. Through different methods, the polymer separation is induced, creating a
new phase (coacervate) [127]. The particle formed can be collected by centrifugation or filtration,
followed by washing, drying, or hardening. This technique can be further divided into complex
or simple coacervation: the simple type is promoted by a change in the medium, which causes a
desolvation in the coating material; while in the complex type, there is a mutual neutralization of two
polymers with opposite charges that will compose the coating [128]. Among the factors that affect the
particle size, which can vary from 20 to 200 µm, are the stirring rate, the phases’ viscosity, the type and
concentration of the surfactant (if added), and the temperature. It is possible to achieve high entrapment
efficiency and good control of the particle size through coacervation. However, the drawbacks reported
for this technique are particle agglomeration, as well as the high operational cost [129].
Emulsification is a technique continuously applied in the food, pharmaceutical, and cosmetic
industries. Briefly, an emulsion consists of a system formed by at least two immiscible liquids,
generally water and oil, where one of the liquids is dispersed as small spherical droplets in the other,
surrounded by a thin interfacial layer of surfactant molecules. The systems where oil droplets are
dispersed in an aqueous phase are called oil-in-water emulsion (O/W), whereas the systems where
water droplets are dispersed in an oily phase are called water-in-oil emulsion (W/O). The addition
of surfactants in the emulsion system is frequently necessary to obtain a kinetically stable solution.
The emulsion particle diameter normally varies from 0.1 to 100 µm [94]. Emulsions are prepared
through the homogenization of the water and oily phases, together with one or more surfactants,
using different methods, such as high-pressure homogenization, microchannel emulsification,
membrane emulsification and ultrasound, among others. The main advantages of this technique
are the relatively easy preparation and low cost; nevertheless, emulsions may suffer from physical
instability when exposed to diverse storage and processing conditions, which could lead to additional
processing steps or the incorporation of additives to improve stability [130]. Another technique based
on the emulsification process is the emulsification-solvent evaporation, which consists of forming an
emulsion of a polymer solution (coating material) and an active agent in a volatile organic solvent,
followed by evaporation of the solvent, usually under atmospheric conditions [131]. This technique is
a simple method to obtain small droplets with a narrow size distribution. However, the high amounts
of organic solvent used may increase production costs [132].
Considering pharmaceutical applications, liposomes are one of the most researched encapsulation
processes. Such a technique involves the formation of lipid vesicles from aqueous dispersions of
amphiphilic molecules, e.g., polar lipids, which tend to produce bilayer structures. Liposomes are
typically spherical, with sizes varying from nanometre to micrometre range. The vesicles formed may
contain a single or multiple layers of amphiphilic polymolecular membranes [133,134]. The possibility
of encapsulating both lipophilic and hydrophilic compounds, promoting targeted drug delivery, and its
versatility in terms of size and number of layers comprise its main advantages. On the other hand,
the potential toxicity due to organic solvent residues, high cost in large scales, and low stability are
considered the main processing obstacles [135].

4. Microalgae Encapsulation

4.1. Functional Foods

Functional food, in general terms, may be defined as a natural or processed food, which contains
an identified component, in qualitative and quantitative amounts, with a proven and documented
health benefit [136,137]. This concept was created in recent decades, opening a new research field
that is in constant expansion due to consumers’ increasing awareness of the close correlation between
Mar. Drugs 2020, 18, 644 12 of 44

diet and health. Beyond providing nutrients required for the bodily metabolism, it is well-known
that food may play a key role in the prevention and treatment of certain diseases, along with the
improvement of physical and mental well-being [138,139]. Following this trend, safety issues regarding
the consumption of processed foods have also become a concern. National authorities, such as the
Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), have restricted
the use of many synthetic additives in food, e.g., synthetic dyes, due to a growth in cancer development
or allergic reactions [6,51].
Accordingly, there is a great interest in the investigation of natural resources and biologically
active compounds with high nutritional value and functionality to be used as a food ingredient in the
development of novel functional foods [140]. Among these, microalgae are emerging as a valuable and
economically viable alternative, as they represent a rich source of food-grade compounds and almost
an unlimited field of exploration due to their abundant taxonomic diversity [140,141]. A variety of
microalgae biomass has already been successfully applied in the fortification of assorted food products,
such as cookies, bread, pasta, and some dairy goods [142].
On the other hand, the incorporation of nutraceutical compounds into food is more of a challenging
approach. The effectiveness of a bioactive as a health-promoting substance within the food matrix
depends on keeping its functionality intact during food processing and storage; conserving the
characteristics (taste, texture, colour, smell, etc.) and acceptability of the original food; and lastly,
assuring the bioavailability of the active ingredients, which includes sustaining sufficient time of gastric
residence without degradation and appropriate gut permeability [95]. Due to the inherent instability
of most bioactive compounds present in microalgae, the efficacy of this process may be compromised.
Consequently, their incorporation into encapsulation systems seems to be a promising strategy to
deliver microalgae health benefits at boosted levels through functional foods [96].
One of the most explored microalgae concerning encapsulation systems for food applications is
the species Haematococcus pluvialis. Several research groups have investigated the encapsulation of its
extract obtained by different methods or purified compounds, essentially the carotenoid astaxanthin.
A resume of the systems reported in the literature and their main findings are described in Table 1.
Mar. Drugs 2020, 18, 644 13 of 44

Table 1. Literature review of encapsulation systems for food applications of the microalga Haematococcus pluvialis, its extracts, or bioactive compounds.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Stability improvement
Disrupted cells Maillard reaction products Spray dryer Functional food [143]
• Better water dispersibility

• Stability improvement under

Homogenized cells Chitosan Immersion Functional food [144]
different storage conditions.

• Precipitation pressure had a

higher influence on the formed
particle size.
Functional food
Astaxanthin or Polymerpoly(hydroxybutirate- • Higher encapsulation efficiency is
Supercritical fluids (SEDS) and [145,146]
carotenoid extract co-hydroxyvalerat)(PHB) achieved when using higher
pharmaceutical
biomass: dichloromethane ratio
(10 mg mL−1 ) at the carotenoid
extraction step.

Extract oleoresin Capsul Spray-dryer Functional food • Thermal stability improvement. [147]

• After one year of storage at

different light, temperature,
and oxygen conditions exposure,
the microparticles were able to
Vibrating-nozzle
Astaxanthin-enriched oil Sodium alginate and low-methoxyl pectin Functional food preserve the astaxanthin content [148]
extrusion technology
ranging from 38% to 94%,
with the highest result found
when they were kept at
lower temperatures.

• Higher photoprotection was

• Chitosan found for the nanoemulsion
• Nanoemulsion without polymeric coating.
Astaxanthin • Carrageenan Functional food [149]
• Extrusion • Chitosan beads provided higher
• Calcium alginate
protection to astaxanthin than
alginate beads.
Mar. Drugs 2020, 18, 644 14 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• High encapsulation efficiency in
the developed nanofibers,
of around 90% for carotenoids
and PUFAs.
Lipid extract Ulvan-pullulan Electrospinning Functional food • Promising protection of the lipid [51]
fraction of H. pluvialis
encapsulated in a natural matrix
composed of
water-based polysaccharides.

• Poly(ethylene oxide)-4- • Only PCPLC was suitable to

methoxycinnamoylphthaloylchitosan form nanospheres.
(PCPLC) Functional food
Polymeric nanospheres by • Greater improvement of
Astaxanthin and [150]
• Poly(vinylalcohol-co-vinyl-4- solvent displacement astaxanthin thermal stability
pharmaceutical
methoxycinnamate) upon PCPLC nanoencapsulation.
• Ethylcellulose (EC)

• Temperature is the most

influential environmental factor
Functional food in astaxanthin degradation.
Astaxanthin Calcium-Alginate Extrusion and • Encapsulation improved [151]
pharmaceutical astaxanthin thermal stability even
after 21 days of storage at
room temperature.

• Microcapsules with 100% whey

protein exhibited the highest
colour and antioxidant stability.
Gum arabic and whey protein, alone or in
Astaxanthin oleoresin Spray-dryer Functional food • The turbidity retention of the [152]
combination with maltodextrin or inulin
microcapsules in aqueous
dispersions depended on the pH
and the carrier.
Mar. Drugs 2020, 18, 644 15 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• The diameter of oleoresin-loaded
beads showed a strong
dependence with alginate
concentration and
alginate/oleoresin ratio.
• Encapsulation yield was
markedly affected by surfactant
Astaxanthin oleoresin Calcium-Alginate External ionic gelation Functional food [153]
and alginate concentrations.
• The mathematical models
developed can be used to predict
the characteristics of natural
astaxanthin-loaded microcapsules
under different
process conditions.

• Astaxanthin exhibited a high

retention rate in the liposomes
• Soy phosphatidylcholine after 15 days of storage at 4 ◦ C.
Astaxanthin • Cholesterol Liposomes Functional food [154]
• Cholesterol
• Enhancement of the
antioxidant activity.

• No astaxanthin loss and particle

size growth were observed in the
astaxanthin-NLCs-added whey
after the storage time.
• Glyceryl behenate Nanostructured lipid • Stability improvement of the
Beverages
• Oleic acid carriers (NLCs) NLCs in non-pasteurized
Astaxanthin oleoresin (whey and [155]
• Lecithin (melt-emulsification/ CO2 -free beer at low
non-alcoholic beer)
ultrasonication technique) storage temperature.
• The organoleptic quality of
NLCs-added beers was
considered acceptable by
the evaluators.
Mar. Drugs 2020, 18, 644 16 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Emulsions prepared with the
starch-soy protein conjugate as
wall material showed better
• Culled banana resistant starch Functional food physical and electrical stability
Astaxanthin oleoresin • Soy protein isolate Emulsification and compared to the one prepared [156]
pharmaceutical only with soy protein.
• Stability improvement at different
storage temperatures (6, 20,
and 37 ◦ C).

• Stability improvement during

6 months of storage at different
Functional food
temperatures in comparison with
Astaxanthin Poly (l-lactic acid) Supercritical anti-solvent and [157]
free astaxanthin.
pharmaceutical
• Lower degradation rates were
found at lower temperatures.

• The addition of XG significantly

increased emulsion stability in
comparison to emulsions
stabilized by WPI alone.
• Emulsified astaxanthin showed
• Whey protein (WPI) higher stability at lower
Astaxanthin oleoresin • Xanthan gum (XG) Emulsification Functional food temperatures during 15 days [158]
of storage.
• The combination of WPI-XG
reduced the digestion and release
of astaxanthin in comparison to
the emulsion system stabilized by
WPI alone.
Mar. Drugs 2020, 18, 644 17 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Stability improvement and greater
• Whey protein Functional food in vitro release rate compared to
Esterified astaxanthin • Arabic gum Complex coacervation and astaxanthin oleoresin. [159]
pharmaceutical • Enhancement of
astaxanthin bioavailability.

• NLCs were stable for at least

180 days at 4 ◦ C and were capable
• Precirol ATO 5 Nanostructured of protecting astaxanthin
Astaxanthin oleoresin • Stearic acid lipid carriers Functional food antioxidant activity. [160]
(hot homogenization) • NLCs exhibited in vitro capacity
to protect human endothelial cells
from ROS.

• O/W emulsion droplets remained

stable at 25 ◦ C with an
• Sodium dode-cyl sulfate encapsulation efficiency of over
Functional food
• Decaglycerol monolaurate Microchannel 98%, during 15 days
Astaxanthin extract and [161]
emulsification storage period.
• Decaglyc-erol monooleate pharmaceutical
• The emulsification process was
highly dependent on the
emulsifier and extract types used.

• The microencapsulated
• Maltodextrin astaxanthin maintained its
Complex coacervation
Astaxanthin oleoresin • Gelatine Functional food antioxidant activity after spray [162]
followed by spray dryer
drying, with higher values than
vitamin C.

• SC-stabilized nanoemulsions
showed good physicochemical
• Modified lecithin (ML) Nanoemulsion stability (>70%) after 30 days
Functional
Astaxanthin • Sodium caseinate (SC) (high-pressure of storage. [163]
beverages
homogenization) • Astaxanthin bioavailability was
strongly influenced by the
emulsifier used.
Mar. Drugs 2020, 18, 644 18 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• The developed microparticles
demonstrated reasonably good
water activity,
• Blends of milk protein (whey protein surface morphology,
Astaxanthin isolate, or sodium caseinate) Spray dryer Functional food encapsulation efficiency, [164]
• Soluble corn fibre and oxidative stability.
• Reconstituted emulsions showed
good stability similar to the
initial emulsions.

• The selected emulsification

method was able to produce
emulsions with remarkably
• Tween 20 narrow droplet size distributions.
Premix membrane
Astaxanthin • Whey protein isolate Functional food • The astaxanthin emulsion was [165]
emulsification
physically stable over 3 weeks of
storage and it was able to
preserve 70% of the astaxanthin
content during this time.

• Nanoparticles showed more

powerful antioxidant activity than
free astaxanthin, by improving
• Chitosan Functional food the cytoprotective effect and ROS
Astaxanthin • Salmon sperm DNA Co-assembly and scavenging efficiency on [166]
pharmaceutical H2 O2 -induced oxidative cell
damage in Caco-2 cells.
• Enhancement of the cellular
uptake efficiency.
Mar. Drugs 2020, 18, 644 19 of 44

Table 1. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Physicochemical characteristics of
the nanodispersions were
significantly (p < 0.05)
• Arabic gum influenced by the type and
• Xanthan gum Emulsification- solvent chemical structure of the
Astaxanthin • Pectin Functional food [167]
evaporation polysaccharides used.
• Methylcellulose • Nanodispersions produced and
stabilized with Arabic gum
presented the smallest particle
size and highest physical stability.

• Stability and antioxidant activity

improvement under acid
treatment, high temperatures,
• β-lactoglobulin and UV radiation.
Spontaneous
Astaxanthin • Chitosan Functional food • Chitosan coating was capable of [168]
self-assembly
providing a surface barrier to
delay the release and degradation
of astaxanthin in the
gastrointestinal tract.

• Resuspended nanoparticles (NPs)

in water exhibited superior
stability than free oleoresin under
extreme pH, high temperature,
Emulsification-Solvent
Astaxanthin oleoresin Whey protein concentrate Functional food UV radiation, and [169]
evaporation
metal-induced oxidation
• Simulated digestion of NPs
showed high
astaxanthin bioaccessibility.
Mar. Drugs 2020, 18, 644 20 of 44

Astaxanthin, which possesses a singular and recognized antioxidant potential, is present in a

considerable amount in the H. pluvialis cyst cells formed under adverse environmental conditions.
Nevertheless, the pure addition of the whole cells into food may not be applicable; during the cyst
phase, the bioactive compounds are surrounded by a thick cell wall, which could hinder proper release
and bioavailability. Therefore, an extraction method is normally required to achieve cell wall disruption
and obtain the compounds of interest [170]. When the extraction process is concluded, highly sensitive
substances, such as astaxanthin, once protected by the cell wall, are now susceptible to the effects
of light, oxygen and high temperatures, among others, which explains the high number of studies
applying encapsulation for this compound [171].
In Table 1, it is possible to observe that all the studies have used as core substance the already
disrupted H. pluvialis cell; purified astaxanthin, as powder or oleoresin; or an astaxanthin-rich extract,
always obtained through green techniques. The coating materials selected for those encapsulation
systems were majoritarian natural polymers, mainly chitosan, alginate, whey protein, maltodextrin,
and Arabic gum, as they are food-grade and widely used as food additives. Regarding the encapsulation
techniques applied for H. pluvialis and its compounds, the diversity reported in the studies is
clear, including both top-down and bottom-up approaches. Among them, spray drying, extrusion,
and emulsification were preferred by half of the authors for the encapsulation processes.
Despite all the differences found for astaxanthin encapsulation, the protective potential of
this strategy over such a compound is unquestionable. The coating layer was reported to
promote stability improvement under storage at adverse conditions, such as high temperatures,
oxygen exposure, or extreme pH values, while preserving its antioxidant potential. Nonetheless,
greater stability was usually detected when the encapsulation system was stored at low temperatures.
Moreover, the enhancement in astaxanthin bioaccessibility and bioavailability was also investigated,
obtaining positive results in in vitro studies.
The development of an encapsulation system is a complex process; as such, understanding the
influence of the type and concentration of the coating material or the applied technique, on the particle
physicochemical properties is also a significant step to guarantee its functionality when added into a
food matrix. Anarjan and Tan [167], for instance, investigated the emulsification and stabilization ability
of four different polysaccharides, namely Arabic gum, xanthan gum, pectin, and methylcellulose,
in the preparation of water-dispersible astaxanthin nanoparticles. The authors reported that the
physicochemical characteristics of the prepared nanodispersions were significantly influenced by
the type and chemical structures of the polysaccharide used as the coating material, with the one
produced with Arabic gum showing the smallest average particle diameter and highest physical
stability. However, they observed a considerable astaxanthin degradation after 30 days of storage
for all samples, which allowed them to conclude that, generally, the nanodispersions produced with
polysaccharides have a larger average particle size and less physicochemical stability than those
obtained with proteins and small molecule emulsifiers.
The encapsulation of bioactives from the marine microalga Phaeodactylum tricornutum for food
applications was described in two different studies, both using the electrospray/electrospinning
technique. This species is considered a significant source of the carotenoid fucoxanthin and PUFAs,
which have been associated with several health-promoting properties. Nevertheless, the rough algal
extract is not suitable for food fortification due to its distinctive odour, consistency, and low bioactive
concentration. Additionally, such lipophilic bioactives possess an inherent sensitivity to many adverse
environmental conditions, low water solubility, compromised bioavailability, and potential degradation
during digestion.
Seeking to overcome these issues, Koo et al. [172] researched the development of a
fucoxanthin-enriched fraction from P. tricornutum-loaded nanoparticles to improve the application
of this carotenoid into the food industry. The nanoparticles were prepared through homogenization
followed by an electrospray system, firstly using only casein as the coating material, then followed by an
extra layer of chitosan. In vitro simulated digestion studies have demonstrated a better bioaccessibility
Mar. Drugs 2020, 18, 644 21 of 44

of the nanoparticles over the P. tricornutum powder. Such a result was also corroborated by the in vivo
pharmacokinetic assay, where the casein-chitosan nanoparticles exhibited superior bioavailability,
possibly due to increased retention or adsorption to the mucin by the presence of chitosan. In another
study, Papadaki et al. [51] recovered a lipid fraction from P. tricornutum through ultrasound-assisted
extraction using coconut oil as a solvent. Subsequently, the extract was emulsified and encapsulated
in ulvan:pullulan nanofibers by electrospinning. The encapsulation process showed an entrapment
efficiency of 90%, for both carotenoids and PUFAs, in food-grade water-based polysaccharides;
thus, representing a promising strategy for incorporation of lipophilic bioactives from the microalga
P. tricornutum into food matrices.
The cultivation and bioactive extraction optimization of the microalga Dunaliella salina are topics
constantly researched in the literature. As the richest natural source of the carotenoid β-carotene,
the encapsulation of this species is also a trending area when it concerns functional foods.
Techniques that were already investigated comprise calcium alginate beads followed by fluidized
bed drying [173] and spray-drying using a mixture of maltodextrin:Arabic gum [174] or different
combinations of gelatine, maltodextrin and Arabic gum, as coating materials [175]. All the researchers
concluded that encapsulation was able to promote stability improvement in the β-carotene content
naturally present in D. salina; however, they also reinforced that better results can be achieved
through the utilization of lower temperatures in the drying process, with the absence of light and high
temperatures during storage.
The encapsulation of microalgae of the genus Chlorella has been widely investigated for
environmental monitoring, but its use in the food industry has not been fully explored yet. Differing from
what has been published about other microalgae species, Chlorella was considered as a possible
coating material in the encapsulation system of other bioactives. Chlorella vulgaris cells were
investigated as a carrier for the polyphenol curcumin [176] and C. pyrenoidosa cells as a carrier
for the carotenoid lycopene [177], aiming at protecting the core substance while developing an
innovative nutraceutical complex. The encapsulation process in both studies was performed by
adsorption. Results demonstrated an increase in the photostability of curcumin by about 2.5-fold,
and an improvement in the thermal and storage stability of lycopene when loaded into Chlorella cells.
Moreover, the Chlorella–lycopene complex presented higher antioxidant activity when compared to
the same amount of free lycopene at room temperature for 25 days, which might be partly due to the
carrier protection, and partly due to the endogenous antioxidants present in C. pyrenoidosa cells.
The species Chlorella pyrenoidosa was also chosen as the object of study of Wang and Zhang [178],
where they evaluated the extraction and antitumor activity of a polypeptide obtained from this
microalga to further encapsulate through two different techniques, namely complex coacervation and
ionotropic gelation. The antitumor activity was confirmed to have inhibitory activity on human liver
cancer HepG2 cells and encapsulation was carried out as a solution to avoid stomach degradation,
followed by a proper release in the intestinal environment. The in vitro release assay revealed that the
encapsulated C. pyrenoidosa polypeptide was well preserved against gastric enzymatic degradation,
increasing its bioavailability at least two-fold when compared to the non-encapsulated bioactive.
Another microalga that has been investigated for food purposes is the species
Phormidium valderianum. Chatterjee et al. [179] reported the encapsulation process by spray-drying of
an antioxidant-rich fraction of P. valderianum obtained through supercritical carbon dioxide extraction,
aiming at enhancing the storage stability of the extracted compounds. A mixture of maltodextrin:Arabic
gum was selected as wall material and the optimization of the microencapsulation process parameters
was performed to achieve the best yield and biological properties, which were examined by antioxidant
capacity, phenolic content and reducing power. The condition that provided the best response
combination of the analysed parameters was spray-drying at an inlet temperature of 130 ◦ C, with wall
material composition of maltodextrin:Arabic gum (70:30). Additionally, a stability study was also
carried out for 60 days, comparing the IC50 values of the DPPH (2,2-diphenyl-1-picrylhydrazyl)
antioxidant assay of non-encapsulated and encapsulated microalgal extract. As a result, it was
Mar. Drugs 2020, 18, 644 22 of 44

confirmed that the encapsulation process was able to protect the antioxidant compounds for a longer
period, enhancing the antioxidant activity shelf-life by eight-fold.
Similarly, Bonilla-Ahumada et al. [180] investigated the microencapsulation of fresh biomass
from the microalga Tetraselmi chuii by spray-drying, along with the effect of the wall material
(maltodextrin:Arabic gum (60:40), chitosan 3% or gelatine 2%) and processing conditions
(inlet temperature 110, 130, and 150 ◦ C) on the preservation of β-carotene and other antioxidant
compounds present in this species. The work reported preservation of 80–92% of β-carotene and
46–81% of the phenolic compounds in freshly microencapsulated microalga, even after three months
of storage in the dark,at 25 ◦ C, when coated with maltodextrin and spray-dried at 130 ◦ C. Moreover,
the authors emphasized the advantage of using spray-drying regarding algal biomass, as it is capable
of protecting unstable metabolites, as well as facilitating the transport and further incorporation into
food products.
The encapsulation process is also widely employed for microalgae of the genus Arthrospira,
focusing on improving several challenges involved in the incorporation of its biomass/powder, extracts,
or compounds into functional foods. A compilation of published studies can be found in Table 2.
The species A. platensis, the main representative of this group, is acknowledged as a pronounced
protein source and rich in many essential nutrients for the human diet. The fortification of different
food products with whole A. platensis biomass has already been explored by many authors seeking
to increase their nutritional content and functionality, i.e., antioxidant potential [181–183]. However,
encapsulation may provide not only a protective layer for stability enhancement over processing and
storage conditions, but the possibility to achieve more uniform distribution in the food matrix.
Mar. Drugs 2020, 18, 644 23 of 44

Table 2. Literature review of encapsulation systems for food applications of the microalga Arthrospira, its extracts, or bioactive compounds.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Thermal stability improvement of
phycocyanin during storage when both
• Alginate polymers were used as the coating material.
Phycocyanin • Chitosan Extrusion Functional food • Microparticles were resistant to the [184]
acidic environment.
• Chitosan-coated alginate microparticles
promoted a superior sustained release.

• Phycocyanin microcapsules with alginate

• Maltodextrin 0.6% and maltodextrin 9.4% showed the
Phycocyanin • Alginate Spray dryer Functional food highest phycocyanin content, [185]
antioxidant activity, encapsulation efficiency,
and blue intensity.

• Optimized conditions of C-phycocyanin

ultrasonic encapsulation exhibited 98%
Ultrasonic and entrapment efficiency when produced with
Phycocyanin Calcium-Alginate Functional food [186]
extrusion techniques 3% sodium alginate, 2.5% calcium chloride,
and 1 mg/mL of
C-phycocyanin concentration.

• Liposomes subjected to homogenization

presented a more uniform morphology and
Purified soybean higher entrapment efficiency than the
A. platensis biomass Liposomes Functional food [187]
phosphatidylcholine sonicated ones, demonstrating a possible
strategy for A. platensis- loaded
liposomes preparation.

• Liposomes allowed the protection of

Functional food phenolic compounds during the digestion
Phenolic extracts Rice and soybean lecithin Liposomes and gastric step, allowing the bioactive [188]
pharmaceutical compounds to be released in the
small intestine.
Mar. Drugs 2020, 18, 644 24 of 44

Table 2. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Microencapsulated A. platensis presented
higher thermal stability than its free form,
showing better anti-inflammatory activity
without exerting cytotoxicity.
Maltodextrin pure or
A. platensis powder Spray dryer Yoghurt • Yogurts added with encapsulated A. platensis [189]
crosslinked with citric acid
presented a more homogeneous appearance.
• Microencapsulation was able to maintain the
yogurt nutritional profile and improve the
antioxidant activity throughout storage time.

• The incorporation of the carotenoid extract

showed high antioxidant activity,
with carotenoids retention six times higher
Yellow passion fruit Solvent when compared to the nanodispersions
Carotenoid extract Functional food [190]
albedo flour displacement method containing synthetic β-carotene.
• The nanodispersions were able to keep the
carotenoids’ stability for 60 days under
refrigerated storage.

• External gelation beads exhibited a more

Extrusion (internal and uniform and homogeneous morphology,
A. platensis powder Alginate Functional food [191]
external ionic gelation) as well as higher protein content when
compared to internal gelation beads.

• Microencapsulation was able to protect the

biomass antioxidant potential in 37.8% from
the pasta cooking conditions.
Arthorspira biomass Calcium-Alginate Spray dryer Pasta • The pasta properties were affected, but the [192]
overall acceptability index was not
influenced by the addition of
the microspheres.

• Improvement of the antioxidant activity and

A. platensis
Calcium-Alginate Vibrational extrusion 3D printed cookies colour stability of 3D printed cookies after [136]
aqueous extract
processing and storage.
Mar. Drugs 2020, 18, 644 25 of 44

Table 2. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Freeze-dried samples, regardless of the
matrix composition, exhibited the highest
carrying capacity.
• Freeze- • The use of ball co-milling caused a complete
Pure trehalose or and spray-drying degradation of the core compound during
A. platensis extract Functional food [193]
with maltodextrin • Ball milling the applied processing.
• Delivery systems containing maltodextrin
were more effective in preventing thermal
degradation and preserving its
colouring ability.

• Ultra-fine particles showed thermal

Phycocyanin Polyvinyl alcohol Electrospray Functional food resistance up to 216 ◦ C, [194]
preserving phycocyanin antioxidant activity.

• Results showed that the type and amount of

• Glycerol monostearate lipid and surfactant had a significant effect
Ultrasound-assisted
and distearate on the properties of the particle.
Phycocyanin high-shear Functional food [195]
• Medium-chain triglyceride • Formulations prepared with glycerol
homogenization
distearate had the highest stability in terms
of deposition and suspended particles.

• Results indicated that phycocyanin

• Maltodextrin microparticles with 9% maltodextrin and 1%
Phycocyanin • K- carrageenan Spray dryer Functional food κ-carrageenan as coating materials produced [196]
the highest bulk density, particle size,
and encapsulation efficiency.
Mar. Drugs 2020, 18, 644 26 of 44

The addition of microencapsulated A. platensis powder obtained through spray drying into
yoghurt was investigated by Da Silva et al. [189] and compared to a formulation containing the
free microalga. The authors reported that microencapsulation was able to promote higher thermal
stability, showing better anti-inflammatory activity without exerting cytotoxicity. Moreover, the
yoghurts incorporated with encapsulated A. platensis exhibited a more homogeneous appearance,
lighter green colour, and noticeable decrease in the strong odour, whilst, at the same time, maintaining
yoghurt’s nutritional profile and an improved antioxidant activity throughout the storage time.
Recently, Zen et al. [192] developed a functional pasta fortified with A. platensis biomass-loaded
alginate microparticles also through spray-drying. Even though the pasta properties were affected by
the addition of microparticles, the overall acceptability index was not influenced according to sensorial
studies. Most importantly, microencapsulation was able to protect 37.8% of the biomass antioxidant
potential from the pasta cooking conditions.
On the other hand, the addition of A. platensis extracts and the protein-pigment phycocyanin—its
main antioxidant compound—into food suffers from the limitations previously described for
food fortification with natural bioactives. The extract composition obtained from this microalga
is highly dependent on the extraction technique and, mostly, on the type of solvent used.
Aqueous-based extracts are essentially rich in phycocyanin, phenolic compounds, and other
polar substances, while organic-based extracts are rich in chlorophyll, carotenoids, and other
lipophilic compounds [197].
Among all the studies, the encapsulation of isolated phycocyanin was investigated by a
considerable number of authors using different techniques, such as spray drying, extrusion,
and electrospraying. The particles’ properties were analysed and optimized to achieve the best coating
material concentration or composition of two distinct types, with the highest entrapment efficiency,
particle size consistency, and stability. Some authors also explored the stability of encapsulated
phycocyanin, describing thermal stability improvement and resistance to the acidic environment when
alginate and chitosan were used together as coating materials [184] and thermal resistance up to 216 ◦ C
with full preservation of its antioxidant activity when encapsulated with PVA [194]. Concerning the
encapsulation of A. platensis extract, phenolic-rich, carotenoid-rich, aqueous-based, and a commercial
powder extract were evaluated as the core of four different encapsulation systems. The beneficial effects
of encapsulation were confirmed through different outcomes, comprising gastric protection of the
phenolic extracts, high stability of the carotenoids, antioxidant potential over storage, and preservation
of colour stability and antioxidant potential of the encapsulated aqueous-based extract.

4.2. Pharmaceutical
Naturally derived products have served as a vital source of drugs since ancient times.
Nowadays, approximately one-third of the top-selling pharmaceuticals are of natural origins or
their derivatives. Plants and microorganisms represent a practically unlimited source of biochemical
molecules, which may have promising pharmacological activities and therapeutic benefits in the
treatment of diverse diseases [198]. In particular, microalgae have shown their importance in the
discovery of new therapeutic molecules, as well as in the isolation and characterization of already
acknowledged ones [199].
As previously mentioned, the application of natural compounds in therapeutics faces significant
shortcomings and developmental challenges, highlighting their usually poor aqueous solubility,
inherent instability, and low bioavailability [200]. The use of micro/nanoencapsulation has been shown
as a solution by the pharmaceutical industry to address the issues associated with these drawbacks,
where the therapeutic value of biologically active compounds can be drastically improved [198].
Microalgae, as a rich and valuable universe of natural products with proven pharmacological properties,
are assumed to benefit from this strategy. However, the application of these microorganisms in drug
delivery systems for pharmaceutical purposes is still a field to be explored.
Mar. Drugs 2020, 18, 644 27 of 44

Similar to what has been reported for microalgae encapsulation in food applications, the species
H. pluvialis, particularly its main bioactive compound astaxanthin, is the most researched one regarding
disease treatment. There is a vast number of biological activities associated with this carotenoid;
however, studies involving encapsulation strategies only consist of a few examples (Table 3). As can be
observed in Table 3, liposomes and nanoemulsion were the encapsulation techniques chosen by most
of the authors, aiming to improve the biopharmaceutical properties of astaxanthin. Through the results,
it was possible to confirm that most of the astaxanthin’s biological activities are due to its unique
antioxidant potential, which is able to protect against diverse deleterious effects of oxidative stress.
The use of encapsulation, nonetheless, significantly boosted the outcomes. The anti-aging activity
of astaxanthin-rich extract loaded nanofibers was investigated by Nootem et al. [201] and not only was
a strong potential against oxidative stress reported, but the nanofibers also promoted a slower in vitro
release profile and increased the stability of the core compounds in comparison with the free extract.
In another study, Chiu et al. [202] proposed that astaxanthin-loaded liposomes could be beneficial
to lipopolysaccharide (LPS)-induced acute hepatotoxicity, which is expressively related to oxidative
stress. The results indicate that, in fact, encapsulated astaxanthin had its bioavailability and liver cell
uptake enhanced, and that the developed drug delivery demonstrated in-vivo hepatoprotective and
acute anti-inflammatory effects, with even superior results than the one found for the positive control
N-acetylcysteine. Overall, astaxanthin therapeutic properties may profit deeply from drug delivery
systems, presenting enhanced effects without cytotoxicity when compared to the free molecule.
Likewise, bioactive compounds from microalgae of the genus Arthrospira were also considered as
the core substance of encapsulation systems focusing on pharmaceutical applications. The protein
C-phycocyanin was the compound with a major interest in this regard; however, phenolic and free
fatty acid-rich extracts were equally investigated. A resume of a literature review comprising
the encapsulation of Arthrospira bioactives as drug delivery systems can be found in Table 4.
According to these studies, the encapsulation of these microalgae compounds for skin delivery has been
particularly explored. Phycocyanin is correlated with several biological properties, whose therapeutic
applications may be challenged by its molecular features (instability and high molecular weight)
and the gastrointestinal acidic environment. Aiming to overcome these issues, Hardiningtyas et al. [203]
studied the possible transdermal permeation of phycocyanin in a solid-in-oil nanodispersion, which was
successfully achieved; the developed encapsulation system was able to facilitate the accumulation of
phycocyanin in the stratum corneum, followed by its permeation into deeper skin layers.
Mar. Drugs 2020, 18, 644 28 of 44

Table 3. Literature review of encapsulation systems for pharmaceutical applications of bioactives or extracts from Arthrospira species.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• In vitro mucoadhesive study revealed that
• Chitosan Coated liposomes the spray-drying method is advantageous to
Colonic drug delivery
C-Phycocyanin • Xanthan gum (chitosomes)/spray prepare C-phycocyanin-loaded chitosomes [211]
(anti-inflammatory)
and freeze-dryer with excellent mucoadhesive properties for
colonic drug delivery.

• Dimyristoylphosphatidylcholine • The encapsulated extract exhibited higher

Phenolic extract • Soybean asolectin Liposomes Antifusarium antifungal activity (90% vs. 74%). and slower [212]
release profile when compared to its free form.

• In vitro skin permeation studies

demonstrated that the nanodispersion with
• Sucrose laurate Solid-in-oil Transdermal drug low surfactant content was able to facilitate
Phycocyanin • Sucrose erucate [203]
nanodispersion delivery the accumulation of phycocyanin in the
stratum corneum, followed by its permeation
into deeper layers for transdermal delivery.

• In vitro skin permeation studies revealed

that phycocyanin loaded-liposomes
exhibited the best drug accumulation into the
• Cholesterol stratum corneum and in the whole skin,
C-Phycocyanin Topical
• Liposomes with higher amounts than the corresponding [213]
from A. platensis l-phosphatidylcholine anti-inflammatory
free phycocyanin gel.
• Liposomes were able to produce the same
topical anti-inflammatory response as the
free phycocyanin by using half the dose.

• Free fatty acids-loaded nanocarriers

A. platensis • Copper-alginate Ultrasound enhanced topical anti-biofilm growth activity
Anti-biofilm [214]
fatty acids emulsification with low concentrations (about 50%
inhibition after 24 h at 0.1 mg/mL).

• The addition of Arthrospira extract into PCL

nanofibers showed potential to be a novel
Tissue engineering
Aqueous extract Polycaprolactone Electrospinning scaffold on fibroblast culture, improving the [215]
(scaffold)
cells’ attachment rate and their
infiltration depth.
Mar. Drugs 2020, 18, 644 29 of 44

Table 4. Literature review of encapsulation systems for pharmaceutical applications of bioactives from the microalga H. pluvialis.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System
• Astaxanthin extract-loaded CA nanofibers
exhibited potential against oxidative stress of
C. elegans with significant values.
Astaxanthin • In vitro release assay showed a
Cellulose acetate (CA) Electrospinning Antiaging [201]
rich-extract prolonged profiled.
• The stability of the nanofibers was
significantly higher compared to that of the
free extract under accelerated conditions.

• Microparticles formed had a good degree of

roundness, dispersity, encapsulation
efficiency, and pH responsiveness to avoid
gastric degradation.
Astaxanthin Calcium alginate Double emulsification Hepato carcinoma • Cellular studies demonstrated that [204]
encapsulated astaxanthin could inhibit
hepatoma cells (HepG2 cell line) but it had
relatively small or no impact on control
hepatocytes (THLE-2 cell line).

• Astaxanthin strongly reduced lipid damage

Lipoperoxidation when different lipoperoxidation promoters
Astaxanthin Egg-yolk phosphatidylcholine Liposomes [205]
inhibition were added simultaneously to the liposomes.

• Mesenchymal stem cell (MSCs)

Proliferation differentiation results showed that 20 ng/mL
Methoxypolyethyleneglycol- astaxanthin-loaded polymeric micelles
and differentiation of
Astaxanthin polycaprolactone (mPEG-PCL) Micelles (self-assembly) enhanced adipogenesis, chondrogenesis, [206]
human mesenchymal
copolymer and osteogenesis of MSCs by 52%, 106%,
stem cells
and 182%, respectively.

• Hepatoprotective and acute anti-inflammatory

effects of the astaxanthin-loaded liposomes
• Cholesterol
Astaxanthin Liposomes Hepatoprotection were confirmed by in vivo assay, being even [202]
• l-phosphatidylcholine
superior to the positive control
(N-acetylcysteine).
Mar. Drugs 2020, 18, 644 30 of 44

Table 4. Cont.

Encapsulation
Core Substance Coating Material Application Major Findings Reference
Technique/System

• Encapsulated astaxanthin activated more

effectively antioxidant enzymes like
superoxide dismutase, catalase,
and glutathione S-transferase than its
• Cholesterol
Astaxanthin Liposomes Antioxidant free form. [207]
• L-phosphatidylcholine
• Astaxanthin-loaded liposomes presented
higher stability and in vitro bioavailability
improvement when compared to the
free molecule.

• The developed nanoemulsion exhibited good

uniformity dispersion and very low
particle size.
Sublingual • In vitro sublingual permeation studies
Astaxanthin Ascorbyl palmitate Nanoemulsion showed that liposomes, together with this [208]
drug delivery
alternative route, are a promising alternative
to enhance the bioavailability and
therapeutic effect of astaxanthin.

• Encapsulated astaxanthin was able to induce

Nanoemulsion ROS generation and apoptosis through the
Astaxanthin and (spontaneous apoptotic signalling pathway, in the nucleus
Sodium caseinate Anticancer [209]
α-tocopherol emulsification- and cytoplasm, as well as disrupt the
ultrasonication) mitochondrial membrane, in cancer cells.

• The carotenoids in their encapsulated form

exhibited an antioxidant potential higher
than the free extract and 9-fold higher when
compared to ascorbic acid.
Carotenoid-rich Polymeric nanocapsules • The developed nanocapsules suspension
Poly-lactide-co-glycolide Antioxidant [210]
extract by solvent displacement when incorporated into a hydrogel showed a
sustained release profile, with a higher
release percentage when compared to the
same formulation with the free extract.
Mar. Drugs 2020, 18, 644 31 of 44

Concerning the treatment of cutaneous diseases, the anti-inflammatory potential of

phycocyanin-loaded liposomes [213] and the anti-biofilm growth activity of A. platensis fatty acid-loaded
coper-alginate nanocarriers [214] were investigated, exhibiting positive effects due to the combination
of the bioactives and encapsulation systems. In the first work, liposomes improved phycocyanin
accumulation in the whole skin, as well as the anti-inflammatory response, which was confirmed by its
superior results when compared to the free phycocyanin gel; while in the second study, the encapsulated
fatty acids were able to inhibit half of the film formation with a very low dosage in 24 h.
The process of encapsulating an active compound also represents a possibility of enhancing
its time of residence in the body; thus, increasing the cell uptake; or promoting an active targeting,
which could be achieved through particle surface functionalization. In this context, Yang et al. [216]
hypothesized that polysaccharides from A. platensis could be used as a surface decorator of nanomaterials
to prevent plasma protein adsorption, maximize circulation time, and improve their cell-penetrating
abilities, more specifically cancer-targeting ability. Selenium nanoparticles were then prepared and
functionalized with purified A. platensis polysaccharides and its cell uptake, cytotoxicity, and in vitro
anticancer activity were evaluated. Results show that the polysaccharides’ surface significantly
enhanced the cell-penetrating and apoptosis-inducing abilities of the selenium nanoparticles towards
several human cell lines; especially in A375 human melanoma cells, where they were found to
be extremely susceptible to the functionalized particle (IC50 of 7.94 µM). Accordingly, A. platensis
polysaccharides were suggested as a potential enhancer of the anticancer activity of nanomaterials.
Equally focusing on anticancer targeting, the microalgae Chlorella protothecoides and Nannochloropsis
oculata had their lipid extract incorporated into two different encapsulation systems in the study performed
by Karakas et al. [217], aiming at bioactive protection and assessment of the in vitro cytotoxicity activity
in human cancer cells. The nano-microparticles were obtained by electrospray and microemulsification
techniques, using PVA:chitosan or PVA:sodium alginate, and calcium alginate, respectively. Based on the
MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), it was possible to confirm that
the encapsulated microalgae extract exhibited cytotoxicity in the cancer cell lines from brain glioblastoma
and colon colorectal carcinoma, while no effect was observed in healthy cells.
Finally, the encapsulation of Dunaliella salina extract was carried out by Zamani et al. [218],
seeking to develop an oral drug delivery system for gastric protection and release control of the
microalga compounds. Arabic gum-coated magnetic nanoparticles were selected as the encapsulation
system for the extracts obtained at the logarithmic and stationary growth phases; and their properties,
such as release profile, antioxidant and anticancer capacity, were assessed. The authors reported that
both formulations promoted a sustained release of D. salina extract in PBS at pH 4.5 and 7.2, with final
relative release values of 72.41 and 43.51% for logarithmic and stationary phases over 48 h, respectively.
Moreover, the antioxidant and cytotoxic activity of the free and nanoparticulated extract on MCF-7
and HeLa cells indicated that both phases presented strong antioxidant and anticancer effects in a
time and dose-dependent manner. Therefore, it was concluded that the oral delivery of encapsulated
D. salina extract seems to be an effective approach to reduce adverse gastric effects and maintain the
functionality of its compounds.

4.3. Cosmetics
Cosmetics are a class of products aimed at improving the structure, morphology, and appearance
of skin or external parts of the body. A large section of this segment comprises skin topical formulations,
which are composed of excipients and one or more active ingredients. Following the current global trend
for products derived from natural sources, there is a demand for the development of environmentally
sustainable cosmetic products, with less chemical compounds, which could act as cosmeceuticals [219].
The interest in microalgae regarding cosmetics application is relatively recent; these microorganisms
produce metabolites in response to changes in the environment, whose main function is linked to the
cell’s ability to regenerate and self-protect against external adverse conditions. In this context, it is
assumed these compounds could instigate the equivalent effect when applied on the skin. Among the
Mar. Drugs 2020, 18, 644 32 of 44

bioactives extracted from microalgae that can be potentially used in cosmetics formulation are the ones
with pronounced antioxidant activity, such as astaxanthin and C-phycocyanin [220,221].
The skin is the outer organ of the body and therefore acts as the primary barrier against the loss
of endogenous substances, as well as the penetration of external agents into the human body. As it
constitutes an interface with the environment, the skin is considered a target of several exogenous
factors, such as UV radiation, pathogens, pollution, and other toxic compounds. Such factors are
usually associated with excessive production of reactive oxygen species and other free radicals,
which are pro-inflammatory mediators and may induce many deleterious effects, including DNA
damage, oxidative stress, photoaging, and carcinogenesis [221,222]. As such, microalgae bioactives
could play an advantageous role in maintaining the skin health status and in the treatment of some
dermatological issues, such as hyperpigmentation, dehydration, photo-oxidation, photoaging, as well
as protection against skin cancer [219,223].
However, the topical application of natural bioactives may be limited not only by their chemical
instability in terms of product development, but by their poor water solubility, which might restrain
the skin absorption and lead to low bioavailability. Additionally, some bioactives also possess a high
molecular weight, which makes their permeation through the first skin layer, the stratum corneum,
impracticable [224,225]. Given these conditions, it is imperative to develop an appropriate and efficient
delivery system for microalgae bioactives onto the skin, which have already been described by a
few authors. The encapsulation of H. pluvialis in liposomes was performed by Hama et al. [226],
and its protective effect on ultraviolet-induced skin damage through topical application was
investigated. Firstly, the authors analysed and confirmed the powerful in vitro antioxidant activity
of astaxanthin-loaded liposomes, which was followed by an in vivo assay. The topical application
of the developed encapsulation system was then capable of inhibiting UV-induced skin damage,
collagen degradation, and melanin production; hence, showing its potential as a protective formulation
against UV-induced skin disorders.
In another study, Sun et al. [224] developed an astaxanthin non-aqueous nanoemulsion through
a high-pressure homogenization method for topical application, to combine the advantages of an
encapsulation system and non-aqueous emulsions. Results show that the system was able to avoid
astaxanthin degradation, keeping its stability for over 4 weeks at 25 ◦ C. Additionally, when compared
to traditional water-based emulsions, the non-aqueous type could effectively improve astaxanthin
chemical stability against light and high temperatures. In vitro cell assays revealed that the non-aqueous
nanoemulsion had low toxicity and protected the cells against oxidative stress. Moreover, in vitro
permeation studies and skin section histological analyses exhibited the enhanced permeation of
astaxanthin with low systemic absorption and unchanged epidermis, which proved the efficacy and
safety of the astaxanthin-loaded non-aqueous nanoemulsion for topical application of that carotenoid.
Likewise, Hu et al. [223] prepared and optimized astaxanthin-loaded PLGA nanoparticles through
the emulsification-solvent evaporation technique and investigated its cellular uptake, cytotoxicity,
and photodamage protective effect in human keratinocyte (HaCaT) cells. According to the in vitro
study, the optimized nanoparticles exhibited excellent cell viability and cell uptake, as well as low
cytotoxicity. Additionally, the photodamage assay demonstrated that the nanoparticles presented
higher antioxidant activity compared to pure astaxanthin after exposure to UVB radiation and
were able to resist photodamage in the cells by reducing ROS levels and restoring mitochondrial
membrane potential.
The encapsulation of the microalga Arthrospira sp. for cosmetic purposes was interestingly explored
by Byeon et al. [227]. The study aimed at the development of an Arthrospira sp. extract-impregnated
nanofiber patch in a double-layer form, which was supported by a PCL nanofibrous cover matrix;
both prepared through the electrospinning technique. The mechanical stability, cytotoxicity,
water absorption, and extract release profile were assessed by the authors. As result, the patch
was found to be non-cytotoxic in human cell-based tests and it presented more moisture and better
adhesiveness than the patch prepared with only alginate nanofibers, which indicates the Arthrospira sp.
Mar. Drugs 2020, 18, 644 33 of 44

extract enhanced those properties, in addition to its biological effects on the skin. Furthermore, the dry
patch promoted the release of most of the extract onto the skin within 30 min, suggesting its potential
to be an innovative skincare product.
The bioactive phycocyanin for food and pharmaceutical applications reported in this review was
commonly derived from the microalga Arthrospira sp. However, the species Aphanizomenon flos-aquae is
also a rich source of this compound and was the object of study of Castangia et al. [225] for cosmetic
purposes. The authors encapsulated phycocyanin in hyalurosomes, a type of phospholipid vesicle
immobilized with hyaluronan sodium, or alternatively in PEG hyalurosomes, due to the high molecular
weight and consequent low bioavailability of this compound. The skin delivery and the protective
potential against oxidative stress damage of these encapsulation systems were assessed through in vitro
cell-based permeability, biocompatibility, and antioxidant activity assays. The permeation studies
demonstrated that hyalurosomes favoured phycocyanin deposition in the deeper skin layers, mainly
when the permeation promoter PEG was added to the particle surface. Results also show that the
phycocyanin-loaded hyalurosomes were highly biocompatible, with improved phycocyanin antioxidant
activity on stressed human keratinocytes when compared to the free compound, also promoting control
in inflammation and a stimulus in keratinocyte proliferation.

5. Conclusions
The interest in microalgae for industrial applications has been growing in the last decade due to the
vast collection of high value biologically active compounds produced by this group. These bioactives
are associated with several pharmacological properties, which have been demonstrated to promote
beneficial effects for human health. Additionally, consumers’ awareness towards the use of healthier,
sustainable, and safer ingredients stimulate the exploration of new natural resources to be applied in
the food, pharmaceutical, and cosmetic industries.
Considering the physicochemical limitations and technological challenges reported for the
incorporation of bioactives into products, namely high instability, poor aqueous solubility, and low
bioavailability, encapsulation systems appear as an emerging and significant tool to overcome such
issues. Microalgae bioactives can be greatly applicable in several areas, but without proper protection
during processing and storage, as well as without suitable biopharmaceutical properties, the efficacy
of their functionality may be utterly compromised.
In this review, we reported different published encapsulation systems incorporated with microalgae
biomass, their extracts, or isolated bioactives to be applied in functional foods, disease treatment,
and dermo-cosmetics. It was noteworthy that microalgae encapsulation for functional foods was
the most investigated field, possibly due to the remarkable nutritional composition found in these
microorganisms. However, many of the systems developed in this regard have proven their effectiveness
in terms of stability and bioavailability improvement, suggesting they could also be applied to
pharmaceutical or cosmetic purposes after in vitro and in vivo biological activity assessment.
For all the applications, the species Haematococcus pluvialis, followed by Arthrospira platensis,
possess the highest number of studies using encapsulation systems, especially concerning their main
bioactive compounds, astaxanthin and C-phycocyanin, respectively. Such bioactives are recognized by
a strong antioxidant potential, which may provide positive effects in the treatment of several disorders.
Accordingly, astaxanthin and phycocyanin are often considered and investigated as health-promoting
substances in diverse areas, reflecting the number of scientific studies.
Overall, the beneficial effects of encapsulation over microalgae and their metabolites are evident.
Through different encapsulation techniques, the studies have described that the presence of a coating
material could, among others, promote stability enhancement under different storage conditions,
allow suitable bioabsorption and skin permeation, improve physicochemical properties, and control
the compound’s release in the target site. Furthermore, in vivo and in vitro evaluation of diverse
biological activities have demonstrated that the encapsulated bioactives are able to provide a much
higher therapeutic effect than their free form. Therefore, combining the biological properties of
Mar. Drugs 2020, 18, 644 34 of 44

microalgae-derived compounds and the advantages of an encapsulation system seems to be a

promising strategy to develop innovative and healthier products.
At the moment, there are only a few examples of commercial products that claim to contain
encapsulated microalgae bioactives, mostly products based on astaxanthin from H. pluvialis [228,229]
and carotenoids from the microalga D. salina [230]. However, it is expected that as studies on the scaling
up of encapsulation processes and the evaluation of in vivo efficacy and safety progress, more and
more companies will start launching new products.

Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/12/644/s1,

Table S1: Different coating materials used for encapsulation systems and their CAS number.
Author Contributions: M.V.V. Conceptualization, Writing—Original Draft, Writing—Review & Editing;
L.M.P. Writing—Review & Editing, Funding Acquisition P.F. Conceptualization, Writing—Review & Editing,
Funding Acquisition, Supervision. All authors have read and agreed to the published version of the manuscript.
Funding: This work was funded by the project “ENHANCE MICROALGAE: High added-value industrial
opportunities for microalgae in the Atlantic Area” (Interreg Atlantic Area 2014-2020, ERDF, ref. EAPA_338/2016)
and FODIAC: Food for Diabetes and Cognition (H2020 MSCA-RISE-2017, contract number: 778388).
Conflicts of Interest: The authors declare no conflict of interest.

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