Figure 2.
Marine origin polysaccharides categorized by electrostatic nature and carboxylated/sulfated structure.
2.1. Alginates
Alginate is a polysaccharide extracted from brown seaweeds, including
Laminaria hyperborea,
Laminaria digitata,
Laminaria japonica,
Ascophyllum nodosum, and
Macrocystis pyrifera [
29]. It is composed by a sequence of two (1→4)-linked α-
l-guluronate (G) and β-
d-mannuronate (M) monomers (
Figure 2). The proportion of M and G blocks may vary with the type of seaweed that it is extracted from. For example, alginate extracted from
Laminaria digitata and
Ascophyllum nodosum have been shown to have M/G ratios of 1.16 and 1.82, respectively [
30]. Alginate is biocompatible, has low toxicity and high bioavailability as well. These are the main advantages that make alginate one of the biopolymers with the widest biomedical applicability. One of the most common applications of alginate is their use as an excipient in DDSs, namely acting as a stabilizer agent in pharmaceutical formulations [
31]. Alginate has carboxyl groups which are charged at pH values higher than 3–4, making alginate soluble at neutral and alkaline conditions [
32]. Such pH sensitivity promotes greater protection for drugs with preferential absorption in the intestinal tract: the acidic environment of the stomach does not disturb the stability of the alginate carrier, whereas in the intestine (where the pH is alkaline) the solubility of this biopolymer—as well as the drug release—is promoted [
33]. Thus, solubility and pH sensitivity make alginate a good biomaterial for the construction of DDSs [
34].
Alginate is widely used for its biocompatibility, low toxicity, high bioavailability, lower extraction and purification costs as compared with other biopolymers, and for the capability to be processed in the form of hydrogel matrices, beads and particles [
12,
35,
36,
37]. Alginate is also used as an excipient in pharmaceutical tablets to promote greater protection and stabilization of the drug. Sodium alginate is the type of alginate mainly used in the pharmaceutical industry in the manufacture of tablets, especially when the drug is not soluble in water. Sodium alginate may be used for the purpose of extending the drug release [
31]. Studies using tablets containing ibuprofen demonstrated that it is possible to control the absorption ratio of the tablets. By using sodium alginate with different chemical structure and degree of viscosity, Sirkia
et al. obtained carriers that triggered either an immediate ibuprofen release or prolonged it, proving that the chemical structure of alginate may influence the release rate of the bioactive agent [
38].
In acidic environments, alginate carboxyl groups are protonated,
i.e., in the –COOH form, being thus uncharged and exhibiting higher viscosity [
32]. This may interfere with the elution of the bioactive agent from the device, thereby limiting drug release when the pH is low [
39,
40,
41]. However, gelling sodium alginate with Ca
2+ ions can solve pH dependent limitations related to the hydration, dilation and erosion of the carrier. Alginate has the ability of cross-linking with Ca
2+ ions through an ionotropic gelation process, usually above pH 6 [
42]. Ca
2+ is not the only ion capable of promoting ionotropic gelation of alginate: Ba
2+ or Zn
2+ ions may also be used for that propose [
43]. Virtually any drug may be entrapped during such mild cross-linking process, and its subsequent release may be dependent on several factors, such as cross-linking extension [
44]. Giunchedi
et al. reported that using sodium alginate, hydroxypropyl methylcellulose (HPMC), calcium gluconate, and ketoprofen as a model drug in the preparation of tablets by direct compression in different combination and ratios may prolong drug release, in particular in tablets with 20% of HPMC [
45]. Alginate hydrogels also have applications in wound healing treatments through the construction of structures used for wound dressings. Several studies show that the bioavailability of drugs encapsulated in alginate hydrogels is greater than if free drug was applied directly at the lesion site, thus increasing the efficacy of healing [
46]. Alginate hydrogels are also used widely in tissue regeneration treatments and cell encapsulation [
47,
48]. Hydrogels obtained from alginate, in particular, present some similar features of the extracellular matrix, thus being appropriate materials to be used in tissue engineering and regenerative medicine applications [
46]. However, it should be noted that the gelling capability of alginate varies with the proportion of G and M groups, with alginates rich in G content yielding higher strength when compared to alginates rich in M groups [
49].
Alginate is also used in the construction of microparticles with the ability to incorporate different bioactive agents, particularly proteins. Alginate microparticles have the capability of retaining large amounts of drug and also promoting protection of the cargo from any proteolytic attack. There are different mechanisms of release of a bioactive agent from the carrier, such as through variations of temperature and pH, and the use of biodegradable materials or enzymatic degradation, among other chemical and physical stimuli-responsive methods [
32,
33,
35,
50]. These parameters are difficult to control and program, since they can vary significantly. However, new release mechanisms from microparticles have been developed, that depended on fully controlled external stimuli, such as ultrasound-triggering. Duarte
et al. developed a type of alginate microparticles which were shown to have perfluorocarbon breakthrough capacity when subjected to vibration by ultrasound waves [
51]. Results showed a disruption of these microparticles after 15 min of exposure, suggesting that such structures are promising DDSs controlled externally by acoustic stimuli (
Figure 3).
Figure 3.
Optical microscope images of alginate microspheres before (
A) and after (
B) ultrasound exposure. Reprinted with permission from [
51], Copyright © 2014 American Chemical Society.
Figure 3.
Optical microscope images of alginate microspheres before (
A) and after (
B) ultrasound exposure. Reprinted with permission from [
51], Copyright © 2014 American Chemical Society.
Over the years, other methods have been developed to fabricate drug delivery particles that promote a better loading efficacy of bioactive substances. Using superhydrophobic surfaces it is possible to produce polymer particles suitable as DDSs. This method allows loading drugs into spherical structures with an encapsulation efficiency close to 100% [
52,
53,
54]. Another strategy to synthesize particles relies on complexation, based on the electrostatic interactions between alginate at neutral and alkaline pH values, bioactive agents and other kinds of naturally occurring polymers, such as the polycation chitosan [
23,
31,
33,
47]. In this matter, alginate complexes have been used to construct DDSs (especially nanoparticles) for gene therapy treatments. The very first systems for the gene delivery were based on genetic material encapsulated within viral vectors. These have several limitations such as the possibility to trigger an immune and inflammatory reactions, infections and mutations. These systems also have high costs of production due to complexity in the processing of viral vectors [
26]. Taking advantage of the capability of natural polymers to form complexes with DNA, safer DDSs could be synthesized to deliver genetic material. The most commonly used polymers in the construction of DNA load vehicles are usually of synthetic origin, for example polyethylenimine (PEI), poly-
l-lysine (PLL), poly(
l-ornithine) and poly(4-hydroxy-
l-proline ester) [
55]. The use of these synthetic materials has allowed the synthesis of complexes via electrostatic interactions between the polymer and the DNA, allowing the creation of a stable complex and the possibility of size adjustment. One of the major limitations of using synthetic materials is their often adverse biological effect in the body. PEI, for example, exhibits elevated levels of cytotoxicity [
56]. In contrast, most natural materials are biocompatibile, biodegradable (in some cases) and show similar capacity to form ionic bonds, therefore providing ensuring good protection for genetic material [
57,
58,
59]. Krebs
et al. developed a calcium phosphate-DNA nanoparticle system incorporated in alginate-based hydrogel for gene delivery to promote bone formation. Results showed a DNA sustained release from the alginate hydrogel around 45% of DNA released after approximately 75 days.
In vivo studies, through the injection of alginate hydrogels containing calcium phosphate nanoparticles and osteoblast-like cells in mice, showed evidence of bone formation [
60].
Taking its anionic nature, alginate can be assembled with polycations as structures other than particles using layer-by-layer (LbL). LbL is used to fabricate ultrathin nanostructured films in a multilayer fashion based on complementary interactions between building blocks, such as polyelectrolytes [
61,
62,
63]. This technique may be useful as a biomimetic approach applied in deconstructing and reconstructing the physiological conditions found in native biological environments, such as the human body [
64]. Polyelectrolyte freestanding films (
i.e., films with a few micrometers in thickness) have been shown to be suitable drug reservoirs of biomolecules, such as growth factors and antibiotics [
65]. This type of films exhibit a good cell adhesion, possibility of cargo entrapment and fast release by variations of electrostatic interactions strength, and also promote a sustained release due to the slow film degradation [
66,
67,
68,
69,
70]. Such multilayer systems can be also used as barriers with controlled mass transporter properties [
71]. The versatility of LbL allows it to be extrapolated to the third dimension to conceive more complex DDSs, such as spherical capsules and tubular structures [
72].
Microcapsules are also typical shapes of alginate processing following different techniques, including emulsion [
73,
74,
75], multiple-phase emulsion [
31,
76] and calcium cross-linked encapsulation [
77]. The ability of alginate to create complexes with other biomaterials by electrostatic interactions, chemical modification or cross-linking can be exploited for building hybrid and more versatile DDSs. Capsules constructed from chitosan/alginate-PEG complexes are reliable models for encapsulating proteins, such as albumin, one of the most common model proteins used in controlled release studies [
78]. The construction of alginate spherical structures with other types of synthetic materials can be a good strategy to extend the versatility of these systems. Using poly(
N-isopropylacrylamide) (PNIPAAm) to take advantage of its thermosensitive properties [
79] in combination with alginate can lead to devices capable of delivering biomolecules with a dual stimuli-responsive dependence (both pH and temperature) [
80]. Studies using indomethacin as a model drug reported that chitosan-alginate-PNIPAAm beads showed lower release rates with decreasing temperatures [
81]. The same occurs when there is a decrease in pH, indicating that it is possible to control the permeability of the particles by controlling both pH and temperature. This approach can lead to the development of DDSs capable of promoting higher control over the release of drugs, proteins and others biomolecules with pharmaceutical interest. Following a similar concept of polymer conjugation, alginate can also undergo complexation with natural polymers, like chitosan, to enhance the absorption and cargo protection in oral delivery, for example, for the administration of insulin [
73,
82].
Alginate may be used in the construction of capsules for cell encapsulation often associated with cytotherapy treatments or simply the creation of cellular microcultures in more complex systems where the use of a conventional bioreactor is not possible. In this context, a new approach to the construction of alginate-based capsules for the incorporation of different types of cells has been presented [
83,
84]. Cells were encapsulated in alginate liquefied particles, coated with multilayer of alternating chitosan and alginate. Along with the cells, poly (lactic acid) microparticles were co-encapsulated to provide anchorage points so that cell survival is promoted. Results demonstrated a high viability of the encapsulated cells and usefulness of these capsules as culture systems. This type of system has wide applicability not only for the cell culture but also in other biomedical applications, since it will allow the encapsulation of different types of cells in combination with other biomolecules such as, for example, growth factors and other cytokines.
2.2. Carrageenans
Carrageenan is a sulfated polysaccharide present in red algae, which structure consists in a linear sequence of alternate residues forming (AB)
n sequence, where A and B are units of galactose residues. These residues may or may not be sulfated. They are linked by alternating α-(1→3) (unit A) and β-(1→4) (unit B) glycosidic bonds (
Figure 2). Unit A is always in
d- conformation, while unit B can be found either in
d- or
l-configuration. The sulfated groups give it a negative charge, which categorizes carrageenans as polyanions [
85]. Carrageenans are classified according to their degree of sulfation: they can be kappa (κ), iota (ι), and lambda (λ), if they have one, two or three sulfate groups respectively. The extraction process is straightforward, consisting in the immersion of the raw material in alkaline solution so that a gel forms. Then follows an extraction step, where the gel is immersed in water heated at 74 °C. Depending on the type of carrageenan and desired degree of purification, it is possible to execute additional purification steps, such as dialysis. The process finishes with filtration, precipitation and drying [
85]. κ and ι types are most frequently extracted from algae of the
Kappaphycus and
Eucheuma genera, while λ type is often extracted from algae belonging to the family Gigantinaceae. The number of sulfated groups influences the gelation capability. Carrageenans κ and ι can form gels in the presence of cations, while the high sulfation degree of λ carrageenan prevent its gelation. Gelation capability has been used in many areas, such as food industries (using carrageenan as an emulsifier and stabilizer), as well as in the cosmetic and pharmaceutical industries [
86].
Contrary to what happens with other biomaterials of marine origin, the use of carrageenan as an excipient in the pharmaceutical industry is not common, thus reports about their applications, characteristics and functions are infrequent. As an example, a study was conducted where two types of carrageenan (κ and ι) were analyzed in terms of compression behavior and their capability of tablet formation [
87]. Results showed that both carrageenans are suitable excipients for controlled release. Carrageenans are also present in various biomedical applications due to their anticoagulant properties [
88], antitumor, immunomodulatory [
89], anti-hyperlipidemic [
90] and antioxidant activities [
91]. They also have a protective activity against bacteria, fungi and some viruses [
92,
93]. Due to the latter, carrageenans have been suggested for possible treatments of respiratory diseases, such as the famous bird flu, and is also being tested for killing other viruses, such as the dengue fever, hepatitis A, HIV [
94] and herpes viruses [
95]. Studies showed that carrageenan, and derivatives of degradation have different levels of toxicity, but do not endanger the health of the patients [
93,
96]. These properties make carrageenan a promising biomaterial for biomedical applications.
The use of carrageenan as an excipient in the manufacture of devices for oral delivery depends mostly on their physicochemical properties, such as water solubility and jellification capability. Carrageenan load capacity depends largely on the sulfation extent, which affects its mechanical properties and its dissolution rate. These factors may affect the release of the cargo, prolonging or accelerating its release [
97]. A greater control over the drug release profile—regardless of other conditions, such as carrageenan type and pH—is possible by association or conjugation with other polymers. The addition of polymers such as hydroxypropyl methylcellulose (HPMC), a temperature sensitive semi-synthetic polymer, can solve problems related to pH erosion and provide higher protection to the drug, thus promoting a sustained release that does not depend on pH [
98]. However, the opposite response may be desired (
i.e., pH-triggered degradation) and, for that, pH responsive polymers may be conjugated. By varying the pH, it is possible to control not only the loading but also the release mechanisms of carrageenan/alginate interpenetrated networks [
99]. The use of stimuli-responsive materials offers another perspective for drug and gene delivery where the carrier may be an active trigger and function as a therapy optimizer. Using temperature-sensitive materials for nanocarriers construction can promote a controlled release at temperatures above 37 °C. Such a system could be helpful in situations as common as a fever. However, it is possible to use other nanocarriers in situations of hyperthermia, where the drug would be available in a localized region [
100,
101].
Carrageenan in the pharmaceutical industry is generally used as a raw material for the construction of DDSs, cell capsules for cell therapies and cartilage regeneration applications [
27,
102]. The use of carrageenan-based hydrogels as a vehicle for the controlled delivery of biomolecules can be a good strategy especially for cargo stabilization Popa
et al. showed that κ-carrageenan hydrogels are adequate environments to encapsulate different types of human cells achieving chondrogenic differentiation [
103]. This system proved to have potential for cartilage regeneration strategies, not only due to the referred differentiation but also because these hydrogels can be easily injectable
in situ and may be used as reservoirs for growth factors [
104]. Carrageenan-based hydrogels, along with other materials of marine origin, have also proved to be suitable good devices for cell encapsulation [
105,
106]. New methods on the production of spherical beads and fibrillar carrageenan/alginate based hydrogel have been developed. Fibrillar hydrogels obtained by wet spinning showed great potential for applications as a cell carrier for cell delivery systems [
107]. Knowing the biological properties of carrageenan, it is hypothesized that carrageenan-based devices are suitable DDSs for the delivery of not only bioactive agents but also of cells for cytotherapies.
Taking advantage of the polyanionic nature of carragenans, they can be combined with polycations via electrostatic interactions. Grenha
et al. developed carrageenan/chitosan nanoparticles through a simple construction method by ionic interactions between polycationic groups of chitosan and polyanionic ones of carrageenan (
Figure 4A) [
108]. This method has the advantage of avoiding the use of organic solvents and harmful cross-linkers. These nanoparticles had a diameter size between 350 and 650 nm. Using albumin as a model protein,
in vitro release tests demonstrated a prolonged release over time, with a 100% of albumin release after three weeks (
Figure 4B). Having a slow release rate is important since it enables the reduction of the encapsulated dose and also provides continuous long-term release without the need for repeated administrations. Cytotoxicity tests demonstrated that these devices present low toxicity. These results are a good indicator that these structures may be feasible for the encapsulation of agents with therapeutic purposes. Carrageenan has also been used in the construction of multilayer structures [
109], microcapsules [
110] and micro/nanoparticles [
111].
Figure 4.
Transmission electron microscopy (TEM) micrograph of chitosan/carrageenan nanoparticles (
A). Ovalbumin release profile from chitosan-carrageenan nanoparticles (
B). Adapted with permission from [
108], Copyright © 2009 Wiley Periodicals, Inc.
Figure 4.
Transmission electron microscopy (TEM) micrograph of chitosan/carrageenan nanoparticles (
A). Ovalbumin release profile from chitosan-carrageenan nanoparticles (
B). Adapted with permission from [
108], Copyright © 2009 Wiley Periodicals, Inc.
2.3. Fucoidans
Fucoidan is a sulfated polysaccharide found in many species of brown algae. It is a polymer chain of (1→3)-linked α-
l-fucopyranosyl residues (
Figure 2), although it is possible to find alternating (1→3) and (1→4)-linked α-
l-fucopyranosyl residues. The structure of fucoidan and its composition depend largely on the extraction source, especially the type of algae. For example, fucoidan extracted from
Fucus vesiculosus is rich in fucose and sulfate, whereas that obtained from
Sargassum stenophyllum contains many more types of residues besides fucose and sulfate, such as galactose, mannose, glucuronic acid, glucose and xylose. A more detailed comparison between several fucoidans and their extraction sources can be found elsewhere [
112]. The extraction can be processed by precipitation using salts or organic solvents, followed by a purification step by chromatography. Recently it was reported that fucoidan has antitumor activity dependent on the degree of sulfation and can inhibit tumor cell proliferation and growth [
113,
114]. However, fucoidan may have inhibitory effects over some cellular functions. Cumashi
et al. demonstrated that fucoidans may exhibit strong antithrombin properties and suppresses tubulogenesis on HUVECs [
22]. Fucoidan has also shown anticoagulant and anti-inflammatory properties, as well as anti-adhesive and antiviral properties [
115,
116].
Like other marine polysaccharides, fucoidan can also be used as a raw material for the construction of DDSs. A typical way of processing fucoidan DDSs is by electrostatic interactions with chitosan, to make microspheres, so-called fucospheres [
117], which have been suggested for burn treatments. Particles with sizes ranging between 367 and 1017 nm were shown to trigger both
in vitro and
in vivo a decrease of the normal burn treatment time due to the increase of regeneration and healing of epithelial tissue [
118]. Taking advantage of the great bioactivity of fucoidan, and the ability to complex with other materials like chitosan, other approaches can be pursued. Huang and Li developed novel chitosan/fucoidan nanoparticles with antioxidant properties for antibiotics delivery (
Figure 5A) [
119]. These nanoparticles presented a spherical morphology and diameter of 200–250 nm. Results showed a highly anti-oxidant effect by reducing concentration of reactive oxygen spices (ROS), using gentamicin as a model drug, release studies showed a controlled release around 99% of gentamicin in 72 h (
Figure 5B). The antioxidant chitosan/fucoidan nanoparticles could thus be effective in delivering antibiotics to airway inflammatory diseases, where the amount of ROS it significantly high. Another approach to take advantage of chitosan/fucoidan interactions as DDSs is to synthesize hydrogels, as described by Nakamura
et al. The authors developed a chitosan/fucoidan microcomplex hydrogel for the delivery of heparin binding growth factors, which showed high affinity with growth factors and were able to promote growth factor activity and also a controlled release [
120].
In vivo studies showed a neovascularization promoted by the growth factors released from the chitosan/fucoidan hydrogel.
Figure 5.
TEM image of chitosan/fucoidan nanoparticles (
A). Gentamicin release kinetics from chitosan/fucoidan particles (
B). Adapted with permission from [
119], Copyright © 2014 distributed under a Creative Commons Attribution License.
Figure 5.
TEM image of chitosan/fucoidan nanoparticles (
A). Gentamicin release kinetics from chitosan/fucoidan particles (
B). Adapted with permission from [
119], Copyright © 2014 distributed under a Creative Commons Attribution License.
Another shape that can be obtained resorting to the polyanionic character of fucoidan are capsules, processed by LbL, particularly fucoidan-chitosan pH sensitive capsules for insulin controlled release [
121]. Pinheiro
et al. used polystyrene nanoparticles with a diameter approximately 100 nm as a template for the deposition of a fucoidan-chitosan multilayered coating [
122]. After construction of the coating, the polystyrene core was removed, being thus possible to incorporate into the capsule numerous bioactive agents. Using PLL as a model molecule, results showed that the release profile was pH dependent and also that the release occurred by diffusion. These results indicate the sensitivity of these particles to pH variations found along the gastro-intestinal tract and the possibility of using these particles as DDSs for oral administration.
2.4. Ulvans
Ulvan is a sulfated polysaccharide extracted from the green algae of the
Ulva and
Enteromorpha genera. Ulvan consists in a polymer chain of different sugar residues like glucose, rhamnose, xylose, glucuronic and iduronic acid with α- and β-(1→4) linkages (
Figure 2). Because of the large number of sugars in its composition, ulvan may exhibit variations in the electronic density and charge distribution, as well as variations of molecular weight. Since it contains rare sugars, ulvan is a natural source for obtaining them upon depolymerization, instead of resorting to chemical synthesis. The extraction process is simple, consisting in adding an organic solvent over the feedstock followed by successive washing steps with hot water, filtration and centrifugation [
123]. Ulvan has several properties of biological interest, such as exhibiting antiviral, antioxidant, antitumor, anticoagulant, anti-hyperlipidemic and immune system enhancing activities. Ulvan also presents low cytotoxicity levels in a wide range of concentrations [
124]. Ulvan is typically used in the food and cosmetic industries, but because of their biological properties, it has a great potential for the development of new DDSs, such as being used as an active principle in pharmacological formulations [
125]. Because of their ability for complexing with metal ions, ulvan can also be used as a chelating agent in the treatment against heavy metal poisoning [
126]. Furthermore, the capacity to process ulvan as nanofibers and membranes has been useful for tissue engineering and regenerative medicine, for example in wound healing treatments [
127].
Ulvan has been used in construction of nanocarriers for biomolecules. Alves
et al. constructed a two-dimensional ulvan-based structure for drug delivery by chemical cross-linking for wound healing [
128]. Using dexamethasone as a model drug, there was a rapid release in the first hour (around 49%), followed by a slower and sustained release, around 75% up to 14 days. Additionally, it is also possible to obtain three-dimensional ulvan-based structures. In this context, ulvan/chitosan particles were produced for the encapsulation and release of dexamethasone [
129]. These particles were incorporated in three-dimensional poly (
d,
l-lactic acid) porous scaffolds for bone tissue regeneration.
In vitro release assays demonstrated a fast release in the first three hours (around 52%), followed by a sustained cumulative release up to 60% in the next 21 days.
Like other marine polysaccharides, ulvan may undergo chemical modifications to synthesize thermostable hydrogels. The addition of other functional groups is also possible so that temperature and light responsive hydrogels are conceived. In this case, ulvan was modified with methacrylate groups to allow jellification by photopolymerization through the irradiation with ultraviolet light [
130]. This is a useful approach to develop cell encapsulation strategies for cytotherapy applications. Ulvan is also used in the construction of membranes, due to electrostatic interactions with other cationic polymers [
131]. Through chemical modification, ulvan and chitosan can also be used as a polymeric component of bone cement, especially due to their mechanical properties [
132].