**2. Marine-Derived Bioactive Compounds against Inflammatory Bowel Diseases (IBD)**

*2.1. Chitosan-Structural Properties and Potential Therapy of IBD*

Chitosan is a linear polysaccharide obtained from deacetylation of chitin, it has a cationic character because of its primary amino groups, which provides it with properties such as controlled drug release, mucoadhesion, in situ gelations, transfection and increased permeation [44,45]. It is an important constituent part of the exoskeleton of arthropods, fungi, and crustaceans, which are the main marine source [46,47]. Among its various properties and applications, those that stand out most are antimicrobial, antioxidant, anticancer and anti-inflammatory [32–37].

The mucoadhesive property of chitosan has been a key point of its application in the treatment of IBD (Table 2) and has also been indicated in a study with patients with IBD [48,49]. For rectal use, this characteristic allows a prolonged local retention time of the drugs. In line with this idea, a chitosan-based hydrogel was able to enhance the efficacy of rectal administration of sulfasalazine (SSZ) in a mice model of ulcerative colitis. The mucoadhesive drug delivery system was more therapeutic than the conventional oral treatment and reduced the plasma concentration of a potentially toxic by-product of the drug [50]. Besides its excellent mucosa adhesion, chitosan polymers are widely employed in targeted drug delivery systems, providing better efficacy results in oral treatments. Pillay et al. were able to develop Stimuli-Synchronized-Matrix (SSM) for colonic delivery, defined by the space, of mesalamine (5-amino-salicylic acid or 5-ASA), the therapeutic metabolite of SSZ, employing chitosan in a polysaccharide matrix coated with an alloy layer [51]. Their SSM was both time and pH-independent, while provided a responsive release of the chemical in the presence of colonic enzymes. These characteristics also allowed the minimization of variations of the plasma concentration and reduced the systemic presence of 5-ASA. A similar approach was used in bioadhesive chitosan pallets and coated beads to increase the topical delivery of 5-ASA [52–54]. Mesalamine has also been loaded in N-succinyl-chitosan microparticles, improving its therapeutic results [54].


**Table 2.** Applications of Chitosan in drug delivery systems for IBD treatment.

Additional research has been conducted to ameliorate the pharmacokinetics of the conventional treatment for IBD. A microgel based on oxidized sodium alginate and watersoluble chitosan demonstrated in vitro potential application as a carrier for mesalamine [55]. A different formulation of chitosan and alginate composite microparticles associated with an enteric coat was capable of effectively delivering 5-ASA and curcumin in a colitis rat model [58]. Another preparation combined a cellulose-derived polymer with pectin and chitosan in matrix tablets of 5-ASA to provide desirable changes in its physicochemical characteristics and drug release profiles [56]. An additional antibacterial effect was reached by preparing mesalamine tablets with a chitosan-ethylenediaminediacetic acid disodium (CH-EDTA) conjugate coating [57]. Research has also been conducted to improve the pharmacokinetics of immunosuppressive drugs. Some strategies, such as loading chitosan modified lipid nanoparticles (NPs) with dexamethasone and preparing azathioprine-loaded chitosan beads have shown promising results in targeted and sensitive drug delivery at the colitis site [59,60]. Dexamethasone microcrystals coated with chitosan, alginate, and an enteric coat multilayers also exhibited significant therapeutic effects in mice [61]. Another corticoid, prednisolone, was loaded with inulin, a naturally occurring polysaccharide, in beads and microparticles coated with calcium (Ca)-alginate core and a chitosan coating. The resulting formulations were tested in vitro and were deemed suitable to be used to deliver substances to the colon [62].

The possibility of producing hydrogels with chitosan to obtain a sustained release of a drug in the intestine is another relevant aspect for its application in the treatment of IBD. A biodegradable and reversible polyelectrolyte complex (PEC) of poly acrylic-acid (PAA) and chitosan was engineered to be used as a swellable hydrogel colonic delivery system for topical treatment of IBD with pentosan polysulfate (PP). However, an enteric coat was still needed to protect the proposed formulation from the gastric environment [63]. Resveratrol, a polyphenol present in red wine with anti-inflammatory properties, was also successfully loaded in nanostructured chitosan-based hydrogel and had its pharmacokinetics improved [64]. Two similar studies demonstrated the use of a chitosan and alginate hydrogel to encapsulate nanoparticles loaded with curcumin and 6-shogaol, a biologic compound found in ginger. The formulations significantly alleviated the colitis symptoms and quickened wound repair in mice (Figure 4) [65,66]. Interesting use of other chitosan hydrogel was in the design of an antibody functionalized nanoparticles-releasing hydrogel. The authors developed nanoparticles prepared with single-chain CD98 antibodies on their surface, for carrying inside of it CD98 small interfering RNAs (siCD98). It was already known that the overexpression of CD98 in the colonic epithelial cells and macrophages was associated with the development and progression of IBD. By using this strategy, the authors were able to downregulate CD98 and efficiently diminished the manifestations of IBD in vitro and in vivo [67]. This idea of using oligonucleotides to inhibit the synthesis of pro-inflammatory molecules has been recently employed to reduce TNF-α production in a mice colitis model. Xu et al. [68] loaded a chitosan-alginate hydrogel with phosphorothioated antisense oligodeoxyribonucleotide of TNF-α (PS-ATNF-α) and reportedly inhibited the molecule at both the protein and mRNA levels [68].

A different approach for targeted delivery of anti-inflammatory substances to macrophages was reported by Chen et al. [69]. The researchers used chitosan associated with alginate and tripolyphosphate (TPP) to form colloidal particles with the drug tylophorine malate (NK007) and to incorporate it inside glucan mannan particles (GMPs). The formulation was capable of specifically delivering the drug to macrophages and effectively cured colitis in the mice model after being administered orally [69]. Chitosan was also used in association with alginate to form gastroprotective microparticles capable of releasing in the small intestine and colon nanoparticles of AvrA, an anti-inflammatory and anti-apoptotic bacterial protein. The authors reported that the formulation diminished clinical and histological scores of inflammations in a colitis model. The encapsulation with alginate and chitosan allowed the drug to be administered orally, instead of transrectally, which was undesired and restricted the delivery to the distal portion of the colon [70]. This association was also applied to develop alginate/chitosan-coated pellets intended for the colon delivery of rutin, a flavonoid with antioxidant and anti-inflammatory effects. The results were promising dissolution profiles and great stability for rutin, which could be a valuable alternative for mild-to-moderate IBD therapy [71].

An analogous approach was applied to the development of chitosan-alginate microspheres as a carrier for icariin, a type of flavonoid, to reduce colonic injury and inflammatory response in rats [72]. Chitosan-alginate microspheres have also been employed to colon deliver a combination of ketoprofen and ascorbic acid, bringing together antiinflammatory and antioxidant properties [81]. Quercetin, a flavonoid, has been incorporated in a chitosan-xanthan microparticle coated tablet, allowing a sustained and targeted delivery to the target [73]. Alike, a hybrid system made liposomes coated with cross-linked chitosan was proposed to deliver quercetin to the intestine [74]. Another strategy to protect a drug against the gastric environment and control its release within the intestine was the development of carboxymethyl starch (CMS)—chitosan monolithic tablets. A study has employed these tablets as a carrier for diamine oxidase and catalase, two enzymes possibly capable of reducing bowel inflammation [75]. A chitosan-derived polymer-enzyme conjugate was developed as a promising option in the treatment of IBD with superoxide dismutase, an antioxidant enzyme [76]. Also for oral delivery, a nanocarrier based on chitosan and fucoidan was loaded with berberine, an alkaloid capable of promoting tightness of the intestinal epithelial tight junction, and revealed promising results [77].

**Figure 4.** Preparation and characterization of 6-shogaol loaded polymeric nanoparticles, (**A**) Schematic illustration of process through which PLGA/PLA-PEG-FA nanoparticles [NPs-PEG-FA] were fabricated using a versatile single-step surface-functionalising technique, (**B**) The morphology of PLGA/PLA-PEG nanoparticles [NPs-PEG] was characterised by transmission electron microscopy [TEM], and their size and zeta potential were measured by dynamic light scattering [DLS] using a Malvern Zetasizer Nano ZS90 Apparatus, and (**C**) The morphology, size, and zeta potential of PLGA/PLA-PEG-FA [NPs-PEG-FA] were characterized. Reprinted from Ref. [66] with permission from Oxford University Press. Copyright © 2017, Oxford University Press. License Number: 5022890402536. Abbreviations: PVA (Polyvinyl alcohol), PLGA (poly(lactic-co-glycolic acid)), PLA (Polylactic acid), PEG (Polyethylene glycol), FA (Folic acid), and NPs (Nanoparticles).

A different design proposed an oil-in-water nanoemulsion coated with a chitosan-based polysaccharide layer film as a nanocarrier for curcumin, an agent with anti-inflammatory and antioxidant properties but lipophilic and unstable in aqueous solutions. The in vitro tests demonstrated that the formulation was capable of protecting the drug from degradation, evidencing its promising use for oral delivery of similar agents (Figure 5) [78]. A multiple stepwise spinning disk processing (SDP) technique was developed to fabricate a drug delivery system based on a composite diclofenac sodium-chitosan-poly(methyl acrylates) nanoparticulate. This approach allowed scale-up manufacturing of the nanoparticulate. Additionally, the drug uptake noticed was three times higher than the control drug solution, with no evident toxicity [79].

**Figure 5.** Schematic representation of air liquid interface of human colon carcinoma cell line (CaCo-2) equivalent epithelium in Transwell system. In the central insert, an enlarged view of the cell monolayer grown on the microporous membrane. On the right, the oral nano-delivery system consisting of oil-in-water (O/W) nanoemulsion coated with a thiolated glycol chitosan. Reprinted from Ref. [78] with permission from Elsevier. Copyright © 2018 Elsevier B.V. License Number: 5022890619360.

A more complex approach employed chitosan nanoparticles as a delivery vector for gene therapy in a colitis mice model. The authors developed a very cost-effective way of constructing a carrier for a plasmid of Single Ig-domain containing IL-1 receptorrelated molecule (SIGIRR), a subtype of the IL-1R family, capable of attenuate colonic tissue inflammation as a result of the inhibition of TLR4/NF-κB overactivation. The study suggested a possible new gene therapy for IBD [80]. Along with the possibility of improving the pharmacokinetics of other compounds, Chitosan oligosaccharide (COS), the major degradation product of chitosan, has also been shown biological activity [82]. For this reason, it has been pointed out as a potential compound to be used in colitis-associated colorectal cancer (CRC) chemoprevention, due to its activation of AMP-activated protein kinase (AMPK) and inhibition of the NF-κB and mTOR signaling pathways in intestinal epithelial cells (IEC) [83–86].

Another relevant application of chitosan is in tissue engineering. Its antimicrobial effect and biodegradability have made it possible for the compound to be used as a bioscaffold for colorectal tissue engineering. A study has shown that a 3-layer chitosan hydrogel patch revealed good wound healing, effective regulation of the inflammation, and an integral regeneration of the colonic wall, along with the smooth cell layer. The latter was achievable due to the soft gel layers on each side, which ensured the colonization of cells and the formation of neo-tissue [87]. In a different approach, an association of chitosan-based hydrogel and stromal vascular fraction from adipose tissue has successfully replaced a circular colonic wall section [88]. Chitosan grafts could be useful for severe cases of IBD that require surgical intervention, such as those on which cancer develops.

#### *2.2. Hyaluronic Acid-Physicochemical Attributes and Potential Therapy of IBD*

Hyaluronic acid (HA) is the only GAG that is not sulfated and not bound to proteins and it's formed by units of disaccharides N-acetyl-D-glucosamine (GalNAc) and D-glucuronic acid (GlcA) [89,90]. HA is a crucial component of the extracellular matrix and performs several functions like cell signaling mediation, morphogenesis, damage repair, and matrix organization [91,92]. HA is found in almost all tissue in humans and also in other vertebrates, can be extracted from marine sources like a shark, stingray, eyeball, liver of swordfish, mollusk bivalves, and tuna [93–96]. It has numerous applications in biotechnology, regenerative medicine, drug delivery vehicles, development of new biomaterials, and other biomedicals and pharmaceutical applications by cause of their properties as biocompatibility, viscoelasticity, lubricity, and immunostimulatory [93,97,98]. HA has been studied as a potential therapeutic agent against several diseases due to its antioxidant, anti-inflammatory, and anticoagulant biological activities [27–31].

HA has potentially useful biological activities against IBD, and studies have shown that sodium hyaluronate, a derivative of HA, has beneficial effects in the treatment for IBD. To investigate whether supplementation of the lining of the colon mucosa with sodium hyaluronate may be a possible and effective alternative treatment for IBD, a clinical study recruited 21 individuals with distal UC and applied 60 mL of sodium hyaluronate gel (IBD98E) once a day for 28 days. 38.1% of patients achieved clinical remission and 47.6% achieved endoscopic remission, showing that the local application of IBD98E enhances endoscopic and clinical outcomes in subjects with active distal UC [99]. However, this study did not have a placebo-controlled group, therefore, the results need to be analyzed with caution. Another study demonstrated the improvement of mucosal healing by quickening intestinal epithelial repair with HA therapy in vitro and in vivo [100]. HA can also be an option to improve the effectiveness of conventional treatments like mesalamine (5-ASA), combined therapy with HA and 5-ASA was capable to accelerate wound repair and diminish inflammatory reaction in rat colitis [101].

Another potential application of HA was reported in a novel study based on the synthesis of methylcellulose (MC) and HA-coated thermo-responsive hydrogel for successful targeted rectal delivery against IBD [102]. The gelling behavior of the hydrogel was improved due to the in situ gelling capability of HA [103]. Moreover, other remarkable features of HA like the slow and prolonged controlled release, non-toxic, stability, and entero-protection were also observed in the mice intestinal model via applicating final formulated hydrogel. It was evident that the HA-MC hydrogel can be employed in safe and inexpensive systems for rectal delivery of substances in IBD [104]. IBD is a risk factor for the development of colorectal cancer and some studies also pointed out applications of HA in these cancer treatments [105–108]. In the last years, nanoparticles (NPs) have been identified with promising strategies for the diagnosis and treatment of many diseases. In comparison to more traditional approaches, they offer advantages such as nanometer-scale dimension, controlled drug release, targeted drug delivery capacity, lower plasma concentrations, and lessen adverse effects [109–111]. Several pieces of research with NPs have been conducted to try to overcome the biggest problems with oral drug administration such as loss of stability in GIT, systemic absorption, risk of side effects, difficulty in transporting sufficient quantities of active drugs, and transporting them to specific target sites [111,112]. In this regard, NPs have been an innovative approach for IBD treatment, and the results have shown that they are much more effective than traditional drug formulation (Figure 6) [111].

Lysine-proline-valine (KPV) is a tripeptide with anti-inflammatory properties, Xiao et al. [113] loaded KPV into HA-functionalized polymeric nanoparticles (HANPs), resulting in an NPs called HA-KPV-NPs. The authors encapsulated the HA-KPV-NPs in a hydrogel (chitosan/alginate) for oral administration against UC in a mouse model and used a group control without HA. The results show that HA-KPV-NPs/Hydrogel system exhibited a much stronger capacity to prevent mucosa injury and downregulate TNF-α compared with group control. Therefore, this study reveals the important role of the HA in the NPs, which was able to penetrate the colitis tissue and allow the KPV to be internalized in the target cells, thereby alleviate UC [113]. In another study that also used a model of UC in mice, HANPs was used to deliver the siRNA of the CD98 transmembrane protein involved in the colon's innate immune responses, siCD98, in association with a robust anti-inflammatory agent, curcumin. The results of the analyzes showed that cell uptake of drugs in groups treated with HANPs was much higher than those treated with NPs without HA, demonstrating that HANPs could be a good alternative for UC-target therapy [114]. The same group reported in previous studies that surface functionalization with HA increases the cellular uptake efficiency of NPs as a consequence of interactions mediated by receptor [67], which seems to be related to the fact that HA is a ligand of CD44, a membrane glycoprotein, that in UC has increased expression on the surfaces of colon epithelial cells and macrophages [115–117]. Accordingly, Vafaei et al. [118] used HANPs to release budesonide in an in vitro model of inflamed CACO-2 cells, the anti-inflammatory effect with HAMPs was much greater

compared to cells that received the free drug [118]. Similar to HANPs, Li et al. [119] use HA functionalized porous silicon nanoparticles to unite enzyme-responsive hydrogel and pH-responsive polymer to develop a hierarchical structured and programmable responsive AP@PSi-HA@HPMCAS carrier for efficient local delivery of drugs to sites of inflammation in the intestine in IBD treatment via oral administrations. The vehicle with HA exhibited superior therapeutic efficacy and significantly diminished systemic drug exposure [119]. No clinical studies have been found using HANPs to treat IBD, however, due to wide marine availability and the promising results of experimental studies they have great potential for the developing of alternative therapies capable of overcoming the limitations of traditional oral drug formulation.

**Figure 6.** Schematic illustration of orally administered cell-specific nanotherapeutics for IBD. Reprinted from Ref. [111] with permission under the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license. Copyright ©The Author(s) 2016. Published by Baishideng Publishing Group Inc.
