Chitin was isolated in 1811 by French chemist and botanist Henri Braconnot, who found in some fungi an insoluble material in alkaline conditions [4, 5]; chitin was the first polysaccharide identified by man, even before cellulose which was discovered in 1838 by French chemist Anselme Payen [6]. In 1823, Antoine Odier found a similar material described by Braconnot while studying the cuticle of some insects and called it chitin, from the Greek "chiton" which refers to a robe or wrap. In 1824, Children extracted chitin from May bug elytra and found nitrogen by elemental analysis. Payen, Fischer and Leuchs also noted the presence of nitrogen in chitin in 1843. Later, Karrer and Zechmeister carried out experiments with chitin and found the N-acetylglucosamine as its main component. Finally, Meyer and Pankow confirmed the structure of chitin through X-ray diffraction studies in the first half of the twentieth century [7].

Chitin has average molecular weight (MW) between 1.03 × 106 and 2.5 × 106 Da and is a polysaccharide insoluble in water as well as in most organic solvents. Chitin can be dissolved in solutions with high ionic strength as hexafluoroacetone or dichloroethane with mineral

**Figure 1.** Chemical structure of: (a) chitin and (b) cellulose.

acids, and 5% lithium chloride in dimethylacetamide [2]. In 2006, Tamura and coworkers solubilized chitin in saturated methanol with dihydrate calcium chloride [8]. The insolubility of chitin is associated with a high degree of crystallinity due to the formation of different hydrogen bonds between the N-acetyl groups (N-H…O=C) at C-2 and hydroxyl group at C-3 and C-6 with N-acetyl group (O-H…O=C), keeping chitin chains tightly bound. There are three crystalline forms for this polysaccharide, depending on the source of chitin. α-Chitin is the most abundant form and is present in the shell of shellfish and fungi cell walls. In this crystalline form, chitin chains are organized in an antiparallel configuration, allowing an orthorhombic crystal to form and giving rigidity to the polymer. In β-chitin form, the chains are aligned in parallel, as do cellulose chains, forming monoclinic crystals. In this case, there are more intramolecular hydrogen bonds rather than intermolecular interactions. β-Chitin is commonly associated with proteins in squid and diatomaceae and has weak packing. Finally, there is a γ-chitin form, which is a mixture of the α and β forms. γ-Chitin combines the properties of both polymorphisms and swells when in contact with water [9].

## **3. Chitosan**

particularly on the current state of knowledge of chitosan functionalization methods, including carboxymethylation, cross-linking, copolymer grafting, among others. Finally, the most recent advances in the biomedical applications of chitosan and its derivatives, especially tissue engineering, drug delivery systems, gene therapy systems and wound healing will be discussed.

Chitin is a linear polysaccharide composed by units of 2-acetamide-2-deoxy-β-d-glucopyranose linked through β(1, 4) bonds. This polysaccharide has a structure similar to cellulose but instead of having a hydroxyl group at carbon number 2, chitin has the N-acetyl group as can

Chitin was isolated in 1811 by French chemist and botanist Henri Braconnot, who found in some fungi an insoluble material in alkaline conditions [4, 5]; chitin was the first polysaccharide identified by man, even before cellulose which was discovered in 1838 by French chemist Anselme Payen [6]. In 1823, Antoine Odier found a similar material described by Braconnot while studying the cuticle of some insects and called it chitin, from the Greek "chiton" which refers to a robe or wrap. In 1824, Children extracted chitin from May bug elytra and found nitrogen by elemental analysis. Payen, Fischer and Leuchs also noted the presence of nitrogen in chitin in 1843. Later, Karrer and Zechmeister carried out experiments with chitin and found the N-acetylglucosamine as its main component. Finally, Meyer and Pankow confirmed the structure of chitin through X-ray diffraction studies in the first half of

saccharide insoluble in water as well as in most organic solvents. Chitin can be dissolved in solutions with high ionic strength as hexafluoroacetone or dichloroethane with mineral

and 2.5 × 106

Da and is a poly-

**2. Chitin**

be seen in **Figure 1**.

88 Biological Activities and Application of Marine Polysaccharides

the twentieth century [7].

Chitin has average molecular weight (MW) between 1.03 × 106

**Figure 1.** Chemical structure of: (a) chitin and (b) cellulose.

Chitosan is obtained via partial or total chitin deacetylation (**Figure 2**) and can be classified as a copolymer of 2-amino-2-deoxy-β-d-glucopyranose (glucosamine) and 2-acetamide-2 deoxy-β-d-glucopyranose (N-acetylglucosamine). In general, when the content of N-acetyl groups is >50% is considered chitin, while for lower values is considered chitosan. It has nitrogen content of 6.80% or higher and is characterized by molecular weights between 1 × 105 and 5 × 105 Da. The discovery of chitosan is attributed to Rouget, who in 1859 found that when chitin was heated in alkaline medium, a material that was soluble in organic acids was obtained [4]. In 1894, Hoppe-Seyler called this material chitosan; however, only until 1950, its chemical structure was elucidated [5].

As a biopolymer, chitosan has potential biomedical applications, since it is biocompatible and biodegradable. Due to its solubility in acidic aqueous medium, many applications at industrial level can be found for chitosan; its solubility is related to the degree of acetylation, molecular weight, and distribution of the acetyl and amino groups along the chain. Additionally, antimicrobial activity is attributed to chitosan when the amino groups are in cationic form, which means that antimicrobial activity of chitosan is higher at low pH [10]. Chitosan has a

**Figure 2.** Chitin deacetylation process to produce chitosan.

broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria [11]. Due to this property, chitosan is a natural antimicrobial agent with potential application in agriculture, food, biomedical and biotechnology fields [12].

## **4. Global distribution of chitin and chitosan in nature**

Chitin is the second most abundant polysaccharide in nature after cellulose [13], it is widely distributed in fungi, diatomaceae, arthropods and other large number of animals and plants; today, a worldwide production of 1011 tons of chitin per year is estimated [14, 15]. The commercial exploitation of chitin has been mainly focused on products derived from the marine food industry, such as shrimp, crabs, lobsters and squid [16]. Each year 6–8 million tons of waste crab, shrimp and lobster shells are produced globally [17]. The major useful components and its percentage on a dry weight basis in commercial crustacean wastes are as follows: chitin 15–40%, protein 20–40% and minerals like calcium carbonate 20–50%. The production of chitin from seafood is reported in countries such as India, Poland, Japan, China, United States, Norway and Australia [3]. On the other hand, the production of chitosan from fungal mycelia has increased in recent decades; the study of different types of fungi as a source of chitosan have been reported, with yields ranging from 1 to 8%, average molecular weights between 5.6 × 104 and 1.6 × 105 Da and degree of deacetylation (DD) between 41 and 70% [18–20].

## **5. Isolation of chitosan**

The production of chitosan is based on a hydrolysis of the acetamide group as shown in **Figure 2**. When fungi are used to produce chitosan, the alkaline treatment removes the protein and deacetylates chitin simultaneously. When the shells of crustaceans are used as source of chitosan, two pretreatment are required, one to remove traces of organic material and another to remove calcium carbonate. Nowadays, there are chemicals and enzymatic methods to produce chitosan. In this section, both methods will be discussed showing the advantage and disadvantage of each of them.

#### **5.1. Chemical method**

During chemical isolation of chitosan, after obtaining the shells from different sources they are washed, dried and size reduced by grounding into a powder [21]. Then demineralization is carried out using acidic treatment with HCl, H2 SO4 , HNO3 , among others. Subsequently, the deproteination of shells requests an alkaline treatment, which usually is carried out with NaOH. Finally, a deacetylation process of chitin is performed using NaOH again but in a higher concentration, with heating, to produce chitosan. This product is dissolved in acidic medium such as diluted acetic acid, formic acid or lactic acid and finally precipitated with dilute sodium hydroxide [2, 22].

The advantages of chemical production of chitosan include the short processing time and the applicability of this methodology at the industrial scale. However, some disadvantages are found:

#### **5.2. Enzymatic method**

broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria [11]. Due to this property, chitosan is a natural antimicrobial agent with potential applica-

Chitin is the second most abundant polysaccharide in nature after cellulose [13], it is widely distributed in fungi, diatomaceae, arthropods and other large number of animals and plants; today, a worldwide production of 1011 tons of chitin per year is estimated [14, 15]. The commercial exploitation of chitin has been mainly focused on products derived from the marine food industry, such as shrimp, crabs, lobsters and squid [16]. Each year 6–8 million tons of waste crab, shrimp and lobster shells are produced globally [17]. The major useful components and its percentage on a dry weight basis in commercial crustacean wastes are as follows: chitin 15–40%, protein 20–40% and minerals like calcium carbonate 20–50%. The production of chitin from seafood is reported in countries such as India, Poland, Japan, China, United States, Norway and Australia [3]. On the other hand, the production of chitosan from fungal mycelia has increased in recent decades; the study of different types of fungi as a source of chitosan have been reported, with yields ranging from 1 to 8%, average molecular weights between 5.6

Da and degree of deacetylation (DD) between 41 and 70% [18–20].

The production of chitosan is based on a hydrolysis of the acetamide group as shown in **Figure 2**. When fungi are used to produce chitosan, the alkaline treatment removes the protein and deacetylates chitin simultaneously. When the shells of crustaceans are used as source of chitosan, two pretreatment are required, one to remove traces of organic material and another to remove calcium carbonate. Nowadays, there are chemicals and enzymatic methods to produce chitosan. In this section, both methods will be discussed showing the advan-

During chemical isolation of chitosan, after obtaining the shells from different sources they are washed, dried and size reduced by grounding into a powder [21]. Then demineralization

the deproteination of shells requests an alkaline treatment, which usually is carried out with NaOH. Finally, a deacetylation process of chitin is performed using NaOH again but in a higher concentration, with heating, to produce chitosan. This product is dissolved in acidic medium such as diluted acetic acid, formic acid or lactic acid and finally precipitated with

The advantages of chemical production of chitosan include the short processing time and the applicability of this methodology at the industrial scale. However, some disadvantages

SO4

, HNO3

, among others. Subsequently,

tion in agriculture, food, biomedical and biotechnology fields [12].

90 Biological Activities and Application of Marine Polysaccharides

**4. Global distribution of chitin and chitosan in nature**

× 104

and 1.6 × 105

**5.1. Chemical method**

**5. Isolation of chitosan**

tage and disadvantage of each of them.

dilute sodium hydroxide [2, 22].

are found:

is carried out using acidic treatment with HCl, H2

Enzymatic treatments offer an alternative way to extract chitosan from crustacean shells. Lactic acid-producing bacteria have been used for demineralization of crustacean shells instead of acidic treatment. The obtained lactic acid reacts with calcium carbonate yielding calcium lactate, which can be precipitated and removed [23]. For the deproteination of crustacean shells, proteases from bacteria are used. The treatment consists of a fermentation of the crustacean by different species of bacteria such as *Pseudomonas aeruginosa* K-187, *Serratia marcescens* FS-3, and *Bacillus subtilis* [24]. Commercial proteases have previously been used to produce chitosan [25]. Chitin acetyl groups are removed by chitin deacetylase [25]. This enzyme was first found in Mucor rouxii. However, Serratia sp. and Bacillus sp. are bacteria that also produce chitin deacetylase and can be used to generate chitosan [23].

The main advantages of using enzymatic methods are the following:

The disadvantage of this biological method is its high cost, which limits its use only to laboratory scale.

## **6. Physicochemical properties of chitosan**

Polysaccharides were considered a source of metabolic energy at first; however, throughout their existence, they have found many applications, as they are nontoxic, biodegradable and abundant in nature. Polysaccharides can be classified according to their acid-base character; there are neutral polysaccharides such as glycogen, cellulose and dextran. Pectin, alginic acid and agar are examples of acidic polysaccharides. Finally, there are some highly basic polysaccharides such as chitin and chitosan [2].

As mentioned previously, chitosan is a linear polysaccharide that contains copolymers of d-glucosamine and N-acetyl-d-glucosamine linked by β(1, 4) glycosidic bonds. The degree of deacetylation (DD) is defined as the glucosamine/N-acetylglucosamine ratio; in other words, DD is the percentage of glucosamine units present in the copolymer chain. Under acidic pH, the amino groups in the chitosan chain become protonated and the polymer dissolves in aqueous media. The protonation constants p*K*a of chitosan change depending on the molecular weight (MW) and DD; p*K*a of 6.51 and 6.39 were found when MW was 1370 and 60 kDa, respectively. On the other hand, p*K*a value was increased from 6.17 to 6.51 when DD decreased from 94.6 to 73.3% [26].

## **7. Characterization of chitosan**

The molecular weight and deacetylation degree are the most important properties of chitosan and dictate the quality and applications of these biomaterials. **Table 1** summarizes the most common methods used to characterize chitosan.

**Table 1.** Chitosan characterization methods.

## **8. Functionalization of chitosan**

Chitosan is a biomaterial that after chemical modifications can find specific biomedical applications. The presence of amino and hydroxyl groups is considered an interesting functionality to modify or improve desired properties. The chemical reactions like cross-linking, carboxymethylation, etherification, graft copolymerization and esterification are the most common modification carried out with chitosan [41].

#### **8.1. Cross-linking**

A hydrogel is a natural or synthetic polymeric system, where the chains are cross-linked through covalent and noncovalent bonds, resulting in three-dimensional networks (**Figure 3**). These systems are able to swell rapidly, retain large volumes of water, while maintaining their original shape [42].

Physical or chemical chitosan hydrogels can be formed, sensitive to external stimuli such as pH or ionic strength. In an acidic medium, free chitosan amino groups are protonated generating electrostatic repulsion that promotes the swelling of the polymeric material [43]. To carry out the formation of chemical hydrogels, chitosan can use both hydroxyl groups at C-3 and C-6, as well as the amino group at C-2. Due to the nucleophilic character of amino groups, they are used widely to react with the cross-linking agent. Therefore, chitosan chains are typically cross-linked with dialdehydes: glyoxal and glutaraldehyde [29]. **Figure 4** shows the scanning electron microscopy (SEM) for a chitosan hydrogels cross-linked with glyoxal and

**Figure 3.** Three-dimensional representation of the polymeric network in a hydrogel.

60 kDa, respectively. On the other hand, p*K*a value was increased from 6.17 to 6.51 when DD

The molecular weight and deacetylation degree are the most important properties of chitosan and dictate the quality and applications of these biomaterials. **Table 1** summarizes the most

Elemental analysis [29]

Fourier transform infrared (FTIR) [30–33] Nuclear magnetic resonance (NMR) [34–36]

Gel permeation chromatography (GPC) [37, 39, 40]

Chitosan is a biomaterial that after chemical modifications can find specific biomedical applications. The presence of amino and hydroxyl groups is considered an interesting functionality to modify or improve desired properties. The chemical reactions like cross-linking, carboxymethylation, etherification, graft copolymerization and esterification are the most common

A hydrogel is a natural or synthetic polymeric system, where the chains are cross-linked through covalent and noncovalent bonds, resulting in three-dimensional networks (**Figure 3**). These systems are able to swell rapidly, retain large volumes of water, while maintaining their

Physical or chemical chitosan hydrogels can be formed, sensitive to external stimuli such as pH or ionic strength. In an acidic medium, free chitosan amino groups are protonated generating electrostatic repulsion that promotes the swelling of the polymeric material [43]. To carry out the formation of chemical hydrogels, chitosan can use both hydroxyl groups at C-3 and C-6, as well as the amino group at C-2. Due to the nucleophilic character of amino groups, they are used widely to react with the cross-linking agent. Therefore, chitosan chains are typically cross-linked with dialdehydes: glyoxal and glutaraldehyde [29]. **Figure 4** shows the scanning electron microscopy (SEM) for a chitosan hydrogels cross-linked with glyoxal and

decreased from 94.6 to 73.3% [26].

**7. Characterization of chitosan**

92 Biological Activities and Application of Marine Polysaccharides

**8. Functionalization of chitosan**

**Table 1.** Chitosan characterization methods.

modification carried out with chitosan [41].

**8.1. Cross-linking**

original shape [42].

common methods used to characterize chitosan.

**Chitosan property Characterization methods** Deacetylation degree (DD) Potentiometric titration [27, 28]

Molecular weight (MW) Viscometry [37, 38]

glutaric acid. The cross-linking of chitosan with glutaraldehyde and sulfosuccinic acid has been used to generate membranes with a good proton conductivity, which makes them useful in fuel cells [44]. Genipin, a compound extracted from the fruit of Gardenia jasminoides and American Genipa has also been used as cross-linker agent [45]. Bodnar and coworkers reported the preparation of chitosan nano-hydrogels, using dicarboxylic and tricarboxylic acids as nontoxic cross-linking agents, which contribute to the biocompatibility of the material [46]. Succinic, glutaric and adipic acid were used recently to prepare chitosan hydrogels [47]. The degree of cross-linking affects hydrogels features such as pore size, mechanical strength and percent swelling. It is clear that small amounts of cross-linking agent increase the absorption capacity. Increasing the cross-link density reduces the swelling due to decreased mobility

**Figure 4.** SEM of chitosan hydrogels cross-linked with: (a) glyoxal and (b) glutaric acid. From Laboratorio de Polímeros, Universidad del Valle.

of the polymer chains. Chitosan hydrogels are also used as controlled release systems, both as epidermal and intracorporeal implants, because they are able to maintain a constant drug concentration in a particular environment for a prolonged time [48, 49]. Due to the biocompatibility of chitosan, hydrogels are not only used for controlled release of drugs such as insulin [50], but also used to produce hemodialysis membranes, biodegradable sutures, artificial substituents skin burns healing agents, immobilizing enzymes and cells, among others.

#### **8.2. Graft copolymerization**

There are various techniques being used to tailor the surface characteristics of chitosan to introduce or improve desired properties. However, graft copolymerization of synthetic polymers with chitosan is of the most importance. Chitosan and carboxymethyl chitosan have been grafted with metacrylic acid by using ammonium persulfate (APS) as initiator in aqueous media. After grafting, the chitosan derivatives had much improved water solubility (**Figure 5a**) [51, 52]. The copolymerization of aniline with chitosan in the presence of APS has been carried out, producing films and fibers, which have shown protonic conductivity (**Figure 5b**) [53]. Different initiators such as, potassium persulfate (PPS), ceric ammonium nitrate (CAN), thiocarbonationpotassium bromate (TCPB), potassium diperiodatocuprate (III) (PDC), 2,2-azobisisobutyronitrile (AIBN) and ferrous ammonium sulfate (FAS) have been used to initiate grafting copolymerization [54].

Due to its solubility in both water and organic solvents, low toxicity, good biocompatibility and biodegradability, poly(ethylene glycol) (PEG) is a suitable graft-forming polymers. PEG grafted with chitosan have been synthesized using the hydroxyl groups of chitosan (**Figure 5c**). The new material was soluble in water and aqueous solutions of wide pH range [55].

#### **8.3. Carboxymethylation**

The low solubility of chitosan is a disadvantage in many of its potential applications. Thus, the preparation of chitosan derivatives has been carried out to improve chitosan solubility in aqueous media. One modification widely used is the carboxymethylation. The procedure consists in dispersing chitosan in isopropanol and NaOH aqueous solution under magnetic stirring and room temperature. Then, a monochloroacetic acid/isopropanol mixture is added to the suspension [56]. Carboxymethyl-chitosan is an amphoteric polymer, and the solubility depends on pH. Although during the reaction O- and N-carboxymethylation may occur simultaneously (**Figure 6**), under controlled reaction conditions (like temperature and stoichiometry) is possible to yield only one of them [37].

#### **8.4. Etherification**

Hydroxypropyl chitosan was prepared from chitosan and propylene epoxide under alkaline conditions. This functionalization allows to carry out graft copolymerization, for example, maleic acid was grafted onto hydroxypropyl chitosan to improve the antimicrobial applications, showing good inhibition effect against *S. aureus* and *E. coli* [51].

**Figure 5.** Chemical structure of graft copolymers: (a) chitosan-PMMA, (b) chitosan-PANI and (c) chitosan-PEG.

#### **8.5. Esterification**

of the polymer chains. Chitosan hydrogels are also used as controlled release systems, both as epidermal and intracorporeal implants, because they are able to maintain a constant drug concentration in a particular environment for a prolonged time [48, 49]. Due to the biocompatibility of chitosan, hydrogels are not only used for controlled release of drugs such as insulin [50], but also used to produce hemodialysis membranes, biodegradable sutures, artificial substituents skin burns healing agents, immobilizing enzymes and cells, among others.

There are various techniques being used to tailor the surface characteristics of chitosan to introduce or improve desired properties. However, graft copolymerization of synthetic polymers with chitosan is of the most importance. Chitosan and carboxymethyl chitosan have been grafted with metacrylic acid by using ammonium persulfate (APS) as initiator in aqueous media. After grafting, the chitosan derivatives had much improved water solubility (**Figure 5a**) [51, 52]. The copolymerization of aniline with chitosan in the presence of APS has been carried out, producing films and fibers, which have shown protonic conductivity (**Figure 5b**) [53]. Different initiators such as, potassium persulfate (PPS), ceric ammonium nitrate (CAN), thiocarbonationpotassium bromate (TCPB), potassium diperiodatocuprate (III) (PDC), 2,2-azobisisobutyronitrile (AIBN) and ferrous ammonium

Due to its solubility in both water and organic solvents, low toxicity, good biocompatibility and biodegradability, poly(ethylene glycol) (PEG) is a suitable graft-forming polymers. PEG grafted with chitosan have been synthesized using the hydroxyl groups of chitosan (**Figure 5c**). The new material was soluble in water and aqueous solutions of wide pH

The low solubility of chitosan is a disadvantage in many of its potential applications. Thus, the preparation of chitosan derivatives has been carried out to improve chitosan solubility in aqueous media. One modification widely used is the carboxymethylation. The procedure consists in dispersing chitosan in isopropanol and NaOH aqueous solution under magnetic stirring and room temperature. Then, a monochloroacetic acid/isopropanol mixture is added to the suspension [56]. Carboxymethyl-chitosan is an amphoteric polymer, and the solubility depends on pH. Although during the reaction O- and N-carboxymethylation may occur simultaneously (**Figure 6**), under controlled reaction conditions (like temperature and stoi-

Hydroxypropyl chitosan was prepared from chitosan and propylene epoxide under alkaline conditions. This functionalization allows to carry out graft copolymerization, for example, maleic acid was grafted onto hydroxypropyl chitosan to improve the antimicrobial applica-

sulfate (FAS) have been used to initiate grafting copolymerization [54].

**8.2. Graft copolymerization**

94 Biological Activities and Application of Marine Polysaccharides

range [55].

**8.3. Carboxymethylation**

**8.4. Etherification**

chiometry) is possible to yield only one of them [37].

tions, showing good inhibition effect against *S. aureus* and *E. coli* [51].

Other chemical modification that is widely documented in the literature is the esterification of chitosan. Special attention has been paid to the preparation of N,O-acyl chitosan using acetyl chloride in MeSO3 H as solvent (**Figure 7**). Under these conditions, O-acetylated chitosan is the

**Figure 6.** N- and O-carboxymethylation of chitosan.

**Figure 7.** O-acylation of chitosan.

major product compared to the N-acetylated chitosan [57]. It has been shown that acetylation of chitosan substantially improve its antifungal activity [58].

#### **8.6. Phosphorylation**

There are some methods to obtain phosphorylated derivatives of chitosan. These derivatives are important due to their interesting biological and chemical properties. They could exhibit bactericidal and osteoinductive properties. Phosphorylated chitosan can be prepared by heating chitosan with orthophosphoric acid in N,N-dimethylformamide (DMF). Another way to prepare phosphorylated chitosan is by the reaction of chitosan with phosphorous pentoxide in methanesulphonic acid, this method is considered very efficient (**Figure 8**) [59]. The methods described here promote phosphorylation of the hydroxyl groups in carbons 3 and 6 of chitosan.

**Figure 8.** Phosphotylation of chitosan using phosphorous pentoxide.

## **9. Biomedical applications of chitosan**

Biomedical applications of chitosan are related with its most important properties, biocompatibility and biodegradability. Biocompatibility is defined as the ability of a material to be biologically inert in the human body or any other guest while performing certain function (ASTM F2475-11). In other words, biocompatibility is directly related to cytotoxicity that may present the material. The cell culture method is the most simple and widely used way to study both the toxicity and the chitosan-cell interaction. L929 fibroblasts studies supported on chitosan have shown biocompatibility of this biopolymer trough no interference with cell growth, on the contrary, favoring its proliferation [60, 61].

On the other hand, biodegradability is defined as the ability of a material to decompose into low molecular weight molecules like carbon dioxide, methane and water by an enzymatic process (ASTM D-5488-944). Biodegradation of chitosan is related to its depolymerization, oligomers produced are subsequently decomposed to the corresponding monomers glucosamine and N-acetylglucosamine. To carry out this process of depolymerization, nature uses the bacterial enzyme known as chitinase, which is present in the digestive tract of vertebrates and many invertebrates such as fish, lizards, birds and mammals such as pigs. For humans, biodegradation of chitosan is mainly associated with the enzyme lysozyme, widely available in macrophages [62].

#### **9.1. Tissue engineering**

major product compared to the N-acetylated chitosan [57]. It has been shown that acetylation

There are some methods to obtain phosphorylated derivatives of chitosan. These derivatives are important due to their interesting biological and chemical properties. They could exhibit bactericidal and osteoinductive properties. Phosphorylated chitosan can be prepared by heating chitosan with orthophosphoric acid in N,N-dimethylformamide (DMF). Another way to prepare phosphorylated chitosan is by the reaction of chitosan with phosphorous pentoxide in methanesulphonic acid, this method is considered very efficient (**Figure 8**) [59]. The methods described here promote phosphorylation of the hydroxyl groups in carbons 3

of chitosan substantially improve its antifungal activity [58].

**Figure 8.** Phosphotylation of chitosan using phosphorous pentoxide.

**Figure 6.** N- and O-carboxymethylation of chitosan.

96 Biological Activities and Application of Marine Polysaccharides

**8.6. Phosphorylation**

**Figure 7.** O-acylation of chitosan.

and 6 of chitosan.

Tissue engineering involves the use of scaffolds for the formation of new viable tissue for a medical purpose. To generate tissue, it is necessary to use a supporting material including natural and synthetic polymers. Polylactic acid (PLA) and polyglycolic acid (PGA) are commonly used for fibroblasts growth at laboratory scale [63, 64]. However, chitosan is now one of the leading cell culture media due to its biocompatibility and because it accelerates the growth process. In many of these studies, chitosan, PLA and PGA are mixed to form films, porous structures or beads [65].

Electrospinning is the production of fiber using electric field to draw charged threads of polymer solutions. This method allows to produce scaffolds with tissue engineering applications from different polymeric materials. Due to the repulsive forces between ionic groups within polymer backbone in chitosan, it is restricted the formation of continuous fibers during the electrospinning process. However, some fiber of chitosan have been prepared successfully using trifluoroacetic acid, which can destroy the rigid interactions between the chitosan molecules, improving the electrospinning process and the diameter of the chitosan fibers [66, 67]. **Figure 9** shows PLA nanofibers with a chitosan coating produced by coaxial electrospinning. These nanofibers nets are used to grow osteoblasts and promote the regeneration of bone tissue.

#### **9.2. Drug delivery systems**

Like agar, glucomannan and pectin, chitosan with low molecular weight can be used as carrier for solid drug formulations because of their easy bioabsorption, with the advantage that it acts as an antacid, preventing stomach irritation [2]. Chitosan has been studied for colon-targeted delivery of a drug, due to its pH sensitivity and its complete digestion by the colonic bacteria [68].

**Figure 9.** Nanofibers of PLA and chitosan produced by coaxial electrospinning. From Escuela de Ingeniería de Materiales, Universidad del Valle.

#### **9.3. Gene therapy systems**

Delivery systems using DNA as a kind of therapeutic agent are considered gene therapy systems, which are a powerful tool for curing many hereditary diseases and treating acquired diseases such as multigenetic disorders [69]. Chitosan is a cationic polyelectrolyte whose positive charge provides a strong electrostatic interaction with negatively charged DNA and protects it from nuclease degradation [70, 71]. For that reason, chitosan is used for nonviral gene delivery. Nanoparticles of folic acid-chitosan-DNA have been used to improve the targeting and transfection rates in gene therapy. These nanoparticles have lower cytoxicity and good DNA condensation. In this case, folic acid is attached to chitosan for targeting cell membrane with folate receptor overexpressed on human cancer cells [72]. Deoxycholic acid-modified chitosan were prepared and characterized to yield self-aggregates/DNA complexes in aqueous media. This chitosan self-aggregates were used as a delivery carrier for the transfection of genetic material in mammalian cells [69]. Reverse microemulsion was used as a template to fabricate chitosan-alginate coreshell nanoparticles encapsulated with enhanced green fluorescent protein (EGFP)-encoded plasmids. These chitosan-alginate coreshell nanoparticles endocytosed by NIH 3T3 cells causes swelling of transport vesicles which renders gene escaping before entering digestive endolysosomal compartment and promotes gene transfection rate [73, 74].

#### **9.4. Wound healing**

Wound healing is a biological process related with growth and tissue regeneration. The wound healing process has five important stages: homeostasis, inflammation, migration, proliferation and maturation [75]. A number of studies have reported the use of chitosan membranes to produce wound healing with potential application in patients with deep burns, wounds, etc. PVA have been mixed with chitosan for the preparation of composites and blends films. PVA-chitosan membranes have shown an increasing of the mechanical properties [41]. Chitosan films with oleic acid and glycerol at 1% were prepared previously. These chitosan films exhibited suitable morphology, and in vivo studies with Wistar® rats showing that implanted chitosan films were biocompatible and bioabsorbable leaving the tissue healthy. For all this, chitosan films may allow their use for wound healing [76]. Due to the importance of antibacterial resistance in the field of wound care to avoid complications such as infection and delayed wound healing, nanoparticles of silver combine with chitosan has been used as an antibacterial agent [75, 77].

#### **9.5. Antioxidant and antimicrobial properties**

Chitosan is a polysaccharide with antimicrobial properties [78]. The antimicrobial mechanism of chitosan is currently unknown. However, it has been suggested that the polycationic nature below pH 6.5 is a decisive factor. The positive charge in the backbone chain interacts with negatively charged components in microbial cell membranes, altering their barrier properties, and thereby preventing the entry of nutrients or causing the leakage of intracellular contents [79]. Due to its ability to form films, chitosan is employed to produce food packaging as a potential food preservative. However, the limited solubility of chitosan restricts its applicability. At the same time, chitosan have been considered a natural antioxidant [80–82]. Thus, different functionalizations have been carried out to improve both the antimicrobial and antioxidant activities of chitosan.

Functionalization of chitosan with epigallocatechin gallate (EGCG) by a free radicalinduced grafting procedure improves both its antimicrobial and its antioxidant activity [83]. Chitosan antioxidant activity can be enhanced by grafting on it protocatechuic acid which is a natural phenolic antioxidant [84]. Chitosan modified with monomethyl fumaric acid presented antibacterial activity against Gram-positive and Gram-negative bacteria [85]. Other chitosan derivatives with antimicrobial activity included the following: chitosan modified with thioglycolic acid [86] and chitosan with polyethylene glycol diacrylate (PEGDA) [87].

## **10. Summary**

**9.3. Gene therapy systems**

98 Biological Activities and Application of Marine Polysaccharides

Universidad del Valle.

**9.4. Wound healing**

Delivery systems using DNA as a kind of therapeutic agent are considered gene therapy systems, which are a powerful tool for curing many hereditary diseases and treating acquired diseases such as multigenetic disorders [69]. Chitosan is a cationic polyelectrolyte whose positive charge provides a strong electrostatic interaction with negatively charged DNA and protects it from nuclease degradation [70, 71]. For that reason, chitosan is used for nonviral gene delivery. Nanoparticles of folic acid-chitosan-DNA have been used to improve the targeting and transfection rates in gene therapy. These nanoparticles have lower cytoxicity and good DNA condensation. In this case, folic acid is attached to chitosan for targeting cell membrane with folate receptor overexpressed on human cancer cells [72]. Deoxycholic acid-modified chitosan were prepared and characterized to yield self-aggregates/DNA complexes in aqueous media. This chitosan self-aggregates were used as a delivery carrier for the transfection of genetic material in mammalian cells [69]. Reverse microemulsion was used as a template to fabricate chitosan-alginate coreshell nanoparticles encapsulated with enhanced green fluorescent protein (EGFP)-encoded plasmids. These chitosan-alginate coreshell nanoparticles endocytosed by NIH 3T3 cells causes swelling of transport vesicles which renders gene escaping before entering digestive endolysosomal compartment and promotes gene transfection rate [73, 74].

**Figure 9.** Nanofibers of PLA and chitosan produced by coaxial electrospinning. From Escuela de Ingeniería de Materiales,

Wound healing is a biological process related with growth and tissue regeneration. The wound healing process has five important stages: homeostasis, inflammation, migration, Chitin is the second most abundant polysaccharide in nature after cellulose. However, due to its solubility problems, scientific research has focused more on studying its main derivative, chitosan. Chitosan is a compound of great interest due to its biocompatibility and biodegradability. At the same time, antimicrobial and antioxidant properties have been found in chitosan. For all these properties, chitosan has potential application at industrial level as well as biomedical applications. The chemical modifications of chitosan through its functional groups have extended its range of applications, improving its mechanical properties, its solubility, among others. Chitosan is a promising material because in addition to various applications, it is a product that is produced from waste seafood, offsetting the negative impact they may have. For all this, we are about to find chitosan commercially available in many everyday products.

## **Author details**

Gustavo Adolfo Muñoz Ruiz and Hector Fabio Zuluaga Corrales\*

\*Address all correspondence to<EMAIL_ADDRESS>
University of Valle, Cali, Colombia

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among others. Chitosan is a promising material because in addition to various applications, it is a product that is produced from waste seafood, offsetting the negative impact they may have. For all this, we are about to find chitosan commercially available in many everyday products.

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#### **Chitosan: Strategies to Increase and Modulate Drug Release Rate Chitosan: Strategies to Increase and Modulate Drug Release Rate**

David Lucio and María Cristina Martínez-Ohárriz David Lucio and María Cristina Martínez-Ohárriz

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http://dx.doi.org/10.5772/65714

#### **Abstract**

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Chitin is the second most abundant polysaccharide present in nature; however, chitin has more applications when transformed into chitosan (CS). It is biocompatible, biodegradable, mucoadhesive, soluble in acidic-solutions, nontoxic and nonallergenic. The main drawback of chitosan in pharmaceutical procedures is its low solubility in physiological medium. Chitosan shows physicochemical characteristics that allow it to interact with a wide variety of molecules. This is of particular interest when increasing the solubility of poor water-soluble drugs. For this purpose, chitosan can be used in oral, nasal and ocular routes. In order to modulate drug release rate and achieve a proper drug delivery in physiological medium, some parameters can be modified when solid dispersions or nanoparticles (NPs) based on chitosan are being designed. In case of nanoparticles, chitosan can be used as the main component or as a modifying agent. In order to optimize drug loading and drug delivery, response surface methodology (RSM) is an interesting tool usually underestimated in the pharmaceutical field, which allows us to optimize the parameters involved in the process simultaneously and not by different steps, which usually lead to mistakes.

**Keywords:** chitosan, carboxymethyl chitosan, drug delivery, nanoparticles, solid dispersions, porous microstructure, response surface methodology

## **1. Introduction**

Chitin is the second most abundant polysaccharide and the only cationic one present in nature; these characteristics make it suitable and interesting for industrial uses [1]. However, chitin has more applications when transformed into chitosan (CS) by partial deacetylation under alkaline conditions [2].

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chitosan [(1–4)-2-amino-2-deoxy-β-d-glucan] is a linear cationic polysaccharide comprising by N-acetyl glucosamine and glucosamine units (**Figure 1**). Although research is more advanced in the case of cellulose, amino groups provide an important advantage to chitosan due to its ease to be chemically modified, including functional groups that would give new properties for more specific uses [3, 4].

**Figure 1.** Representation of chitosan molecule.

Chitosan are receiving increasing attention in the industrial domain as a consequence of their special properties. It is a biocompatible, biodegradable, mucoadhesive, and transmucosal penetration enhancer, soluble in acidic solutions, nontoxic and nonallergenic [5]. Moreover, because it is generally recognized as safe (GRAS), which together with its abundance and low cost make chitosan a particularly interesting material for its use in the pharmaceutical field [6, 7]. In addition, its antacid and antiulcer activities made chitosan and its derivatives an interesting alternative if gastric irritation wants to be avoided when anti-inflammatory drug are administered [8, 9]. Finally, chitosan is an excellent direct compression adjuvant, an important characteristic for potential pharmaceutical formulations [10].

**Figure 2.** Representation of molecules of O-carboxymethyl chitosan (A), N-carboxymethyl chitosan (B), trimethyl chitosan (C), and N-alkyl chitosan (D).

The main drawback of chitosan in pharmaceutical procedures is its low solubility in aqueous solutions and physiological medium [11, 12]; acetylation degree and molecular weight are the most relevant variables which condition chitosan solubility [13]. However, this problem can be solved, thanks to the easy modification of its primary aliphatic amine group. This fact allows us to obtain water-soluble derivatives such as carboxymethyl chitosan, trimethyl chitosan, and N-alkyl chitosans (**Figure 2**) [14]. Both chitosan and its derivatives have physicochemical characteristics that allow them to interact with a wide variety of drugs. This is of particular interest when trying to increase the solubility of poor water-soluble drugs [15, 16], which is a fundamental strategy to increase low bioavailability of drugs belonging to Groups II (low solubility, high permeability) and IV (low solubility, low permeability) of Biopharmaceutical Classification System (BCS). In this sense, different strategies have been developed to increase the solubility of these drugs, such as particle size reduction, drug fusion and preparation of solid dispersions, nanosuspensions, amorphous states, inclusion complexes, or nanoparticles (NPs).

Chitosan [(1–4)-2-amino-2-deoxy-β-d-glucan] is a linear cationic polysaccharide comprising by N-acetyl glucosamine and glucosamine units (**Figure 1**). Although research is more advanced in the case of cellulose, amino groups provide an important advantage to chitosan due to its ease to be chemically modified, including functional groups that would give new

Chitosan are receiving increasing attention in the industrial domain as a consequence of their special properties. It is a biocompatible, biodegradable, mucoadhesive, and transmucosal penetration enhancer, soluble in acidic solutions, nontoxic and nonallergenic [5]. Moreover, because it is generally recognized as safe (GRAS), which together with its abundance and low cost make chitosan a particularly interesting material for its use in the pharmaceutical field [6, 7]. In addition, its antacid and antiulcer activities made chitosan and its derivatives an interesting alternative if gastric irritation wants to be avoided when anti-inflammatory drug are administered [8, 9]. Finally, chitosan is an excellent direct compression adjuvant, an

**Figure 2.** Representation of molecules of O-carboxymethyl chitosan (A), N-carboxymethyl chitosan (B), trimethyl chito-

The main drawback of chitosan in pharmaceutical procedures is its low solubility in aqueous solutions and physiological medium [11, 12]; acetylation degree and molecular weight are the most relevant variables which condition chitosan solubility [13]. However, this problem can be solved, thanks to the easy modification of its primary aliphatic amine group. This fact allows us to obtain water-soluble derivatives such as carboxymethyl chitosan, trimethyl chitosan, and N-alkyl chitosans (**Figure 2**) [14]. Both chitosan and its derivatives have physicochemical characteristics that allow them to interact with a wide variety of drugs. This is of particular interest when trying to increase the solubility of poor water-soluble drugs [15, 16], which is a

important characteristic for potential pharmaceutical formulations [10].

properties for more specific uses [3, 4].

108 Biological Activities and Application of Marine Polysaccharides

**Figure 1.** Representation of chitosan molecule.

san (C), and N-alkyl chitosan (D).

For this purpose, chitosans can be used in oral, nasal, and ocular routes, being the first one considered as the safest and most advisable way. Drug-controlled release by oral route has been achieved using chitosan in several pharmaceutical dosage forms such as solid dispersions, cross-linked hydrogels or nanoparticles [17, 18]. In case of nanoparticles, chitosan can be used both as modifying agent and as the main component by different methods such as cross-linking, ionic gelation, or solvent evaporation.

As it is well known, drug delivery from polymeric systems is influenced by several factors: polymer molecular weight, deacetylation and substitution degree of polysaccharides, nanoparticle size, porous structure, or compression force are some of the factors which directly determine the release rate from a solid matrix. The length and conformation of polysaccharide chains influence the accessibility of chitosan functional groups by drug molecules, which is crucial to establish electrostatic interactions and the subsequent incorporation of drug molecules into these systems.

Furthermore, different preparation methods can lead to different interactions and porous structures when nanoparticles are formed or tablets are prepared by compression [19]. Porous microstructures (pore size distribution, porosity, tortuosity, etc.) strongly affect the penetration of solvent into matrixes, dissolution of drug molecules, and subsequent release of these molecules to the medium. This fact can be observed when the release rate studies were conducted for similar systems but different porous structures as consequence of preparation method.

In these pharmaceutical processes, there are some parameters which can be modified with the typical aims to incorporate the maximum amount of drug into the systems or to achieve a proper drug delivery in physiological medium. In order to optimize these characteristics, design of experiments is an interesting tool usually underestimated in the pharmaceutical field; however, recently, it is being considered a point of interest to maximize the benefits of some processes. Simplifying the process, reducing the economic cost, and improving the characteristics of the final formulation are key features that can make a formulation cross the barrier from laboratory to market. For this purpose, response surface methodology (RSM) has proven to be a suitable tool when trying to optimize a procedure [20]. This strategy is especially interesting when the response to be optimized is influenced by two or more parameters simultaneously or when two different responses must be optimized at the same time.

It is worthy to note that one of the most common mistakes when a response is being optimized is trying to modify each parameter independently. All individual parameters may produce a determined variation in the response depending on the specific value taken by the other influential parameters. For this reason, this parameter modification should be done simultaneously and not individually.

In the past, our group has explored the use of different polymers such as PEG [21], PVP [22], poly(anhydride) [23], chitosans [18], or cyclodextrins [24, 25] to obtain systems, which can increase the bioavailability of different drugs. To improve the properties of these delivery systems, along with the drug of interest, various polymers can be combined to provide improvements in several properties, such as the drug solubility increase and the achievement of a controlled release system at the same time.

The aim of the current chapter was to investigate the possibilities and advantages of chitosanbased system for the oral administration of drugs, which show low bioavailability as consequence of its low solubility in different dosage forms: tablets and nanoparticles. Diflunisal (DF) has been chosen as model molecule (**Figure 3**). It is a drug with analgesic and anti-inflammatory activity, and it has been shown to interact with a high variety of polymers [26].

**Figure 3.** Representation of diflunisal molecule.

## **2. Materials and methods**

#### **2.1. Materials**

Diflunisal (DF) was kindly supplied by Merck Sharp and Dohme (Spain). Chitosan (>375 kDa) was supplied by Sigma-Aldrich with a degree of deacetylation of 75–85%. 2-(Hydroxypropyl) β-cyclodextrin (HPβCD) was purchased from Cyclolab (Hungary). Hydroxypropyl methylcellulose (HPMC) K4M was obtained from Colorcon (Orpington, UK), and sodium alginate (Alg) was supplied by Sigma-Aldrich. The reagents ethanol (Scharlau, PA), ammonium hydroxide, and hydrochloric acid (Panreac, analytical grade) were used as received. All aqueous solutions were prepared with deionized water obtained from a commercial Millipore Elix 3 system (0.1 μS/cm conductivity).

## **2.2. Preparation of solid dispersions**

influential parameters. For this reason, this parameter modification should be done simulta-

In the past, our group has explored the use of different polymers such as PEG [21], PVP [22], poly(anhydride) [23], chitosans [18], or cyclodextrins [24, 25] to obtain systems, which can increase the bioavailability of different drugs. To improve the properties of these delivery systems, along with the drug of interest, various polymers can be combined to provide improvements in several properties, such as the drug solubility increase and the achievement

The aim of the current chapter was to investigate the possibilities and advantages of chitosanbased system for the oral administration of drugs, which show low bioavailability as consequence of its low solubility in different dosage forms: tablets and nanoparticles. Diflunisal (DF) has been chosen as model molecule (**Figure 3**). It is a drug with analgesic and anti-inflamma-

Diflunisal (DF) was kindly supplied by Merck Sharp and Dohme (Spain). Chitosan (>375 kDa) was supplied by Sigma-Aldrich with a degree of deacetylation of 75–85%. 2-(Hydroxypropyl) β-cyclodextrin (HPβCD) was purchased from Cyclolab (Hungary). Hydroxypropyl methylcellulose (HPMC) K4M was obtained from Colorcon (Orpington, UK), and sodium alginate (Alg) was supplied by Sigma-Aldrich. The reagents ethanol (Scharlau, PA), ammonium hydroxide, and hydrochloric acid (Panreac, analytical grade) were used as received. All aqueous solutions were prepared with deionized water obtained from a commercial Millipore

tory activity, and it has been shown to interact with a high variety of polymers [26].

neously and not individually.

of a controlled release system at the same time.

110 Biological Activities and Application of Marine Polysaccharides

**Figure 3.** Representation of diflunisal molecule.

Elix 3 system (0.1 μS/cm conductivity).

**2. Materials and methods**

**2.1. Materials**

Diflunisal-chitosan solid dispersions were prepared by kneading at 20:80 drug/polymer ratio. Chitosan was wetted in a mortar with a minimum volume of an ethanol/water solution (50%, v/v) and kneaded with a pestle, while diflunisal was added until a dense paste was formed.

#### **2.3. Preparation of nanoparticles**

Chitosan-based nanoparticles can be easily formed via ionic gelation due to the interaction between the positive charges of chitosan amine groups and negative charges of sulfate ions. In order to incorporate the maximum amount of drug into nanoparticles, inclusion complexes with cyclodextrins were previously formed by a coevaporation method.

Chitosan- and drug-cyclodextrin complexes were dissolved in water and added over an aqueous solution containing sodium sulfate (SS). After the immediate formation by ionic gelation, nanoparticles were centrifugated at 20,000 × *g*, frozen, and then freeze-dryed.

#### **2.4. Optimization of nanoparticles: response surface methodology**

The effective parameters in the NP formation process, including CS, sodium sulfate, and DF-HPβCD complex concentrations were optimized using response surface methodology. Statgraphics Centurion XVI.I software was used in order to find the best combination of factors which minimize nanoparticle diameter and polydispersity index (PDI) simultaneously. Central composite design (CCD) was chosen due to its orthogonal and rotable properties. It is also an appropriate design of experiments when a process is influenced by more than two factors at the same time because of the reduction of the experiment number:

$$\text{Number of experiments} = \text{2"} + \text{ 2:n + central point realizations} \tag{1}$$

where *n* is the number of factors involved in the process optimization.

#### **2.5. Characterization techniques**

#### *2.5.1. X-ray diffractometry (XRD)*

X-ray powder diffraction patterns were collected on a X Bruker axs D8 Advance diffractometer (Karlsruhe, Germany) using a CuKα radiation.

#### *2.5.2. Scanning electron microscopy*

The shape and surface morphology were examined by scanning electron microscopy (SEM) (Zeiss DSM 940 A apparatus) provided with a digital image capture system (DISS de Point Electronic GmBh).

#### *2.5.3. Transmission electron microscopy (TEM)*

The morphology and size of nanoparticles in water solution were analyzed using a transmission electron microscope (TEM). Samples were analyzed using a LIBRA 120 energy-filtering TEM (Zeiss) operated at 80 kV.

#### *2.5.4. Dynamic light scattering*

The size distribution of nanoparticles was determined by dynamic light scattering (DLS) using a DynaPro photon correlation spectrometer at 25.0 ± 0.1°C. The intensity of size distributions, expressed in terms of the hydrodynamic radius (*R*h), was calculated by the Stokes-Einstein equation:

$$R\_{\text{h}} = \frac{kT}{6\pi\eta\_{0}D\_{0}}\tag{2}$$

#### *2.5.5. Zeta potential*

The zeta potential of nanoparticles was measured by electrophoretic laser Doppler anemometry using a Zetamaster analyzer system (Malvern Instruments, UK).

#### **2.6. Dissolution rate studies**

The in vitro dissolution studies of pure drug and tablets including solid dispersions were performed using tablets containing 150 mg of diflunisal-chitosan solid dispersions. These dispersions were pressed on an instrumented single-punch tablet press (Kilian SP300, IMA, Germany) using round flat-faced punches (7 mm diameter and 3.15 mm thickness). HPMC and sodium alginate were added directly before the compression process.

The dissolution rate assays were performed in a SOTAX AT 7smart (SOTAX, Basel, Switzerland) tablets, or NPs were placed in 900 mL of PBS (Phosphate Buffer Solution) 7.4 medium at 37.0°C and stirred at 50 rpm.

## **3. Results and discussion**

#### **3.1. Optimization of nanoparticle formation process**

Nanoparticles were easily formed via ionic gelation due to the interactions between positive charges of chitosan and negative charges of sodium sulfate.

Optimization of particle size was carried out using response surface methodology and central composite design of experiments. In previous experiments, several characteristics of the process were studied. Nanoparticles were formed using chitosan of high, medium, and low molecular weight; high molecular weight was the most suitable material because the chain length is longer, and this fact facilitates folding and therefore the nanoparticle formation when contact to the negative charges of sodium sulfate. The volume of solutions employed and stirring rate were shown to be non-influential factors in the process. **Table 1** shows factors to optimize particle size and polydispersity index responses.

*2.5.3. Transmission electron microscopy (TEM)*

112 Biological Activities and Application of Marine Polysaccharides

TEM (Zeiss) operated at 80 kV.

*2.5.4. Dynamic light scattering*

equation:

*2.5.5. Zeta potential*

**2.6. Dissolution rate studies**

37.0°C and stirred at 50 rpm.

**3. Results and discussion**

**3.1. Optimization of nanoparticle formation process**

charges of chitosan and negative charges of sodium sulfate.

The morphology and size of nanoparticles in water solution were analyzed using a transmission electron microscope (TEM). Samples were analyzed using a LIBRA 120 energy-filtering

The size distribution of nanoparticles was determined by dynamic light scattering (DLS) using a DynaPro photon correlation spectrometer at 25.0 ± 0.1°C. The intensity of size distributions, expressed in terms of the hydrodynamic radius (*R*h), was calculated by the Stokes-Einstein

> 0 0 6 *kT R* ph

The zeta potential of nanoparticles was measured by electrophoretic laser Doppler anemom-

The in vitro dissolution studies of pure drug and tablets including solid dispersions were performed using tablets containing 150 mg of diflunisal-chitosan solid dispersions. These dispersions were pressed on an instrumented single-punch tablet press (Kilian SP300, IMA, Germany) using round flat-faced punches (7 mm diameter and 3.15 mm thickness). HPMC

The dissolution rate assays were performed in a SOTAX AT 7smart (SOTAX, Basel, Switzerland) tablets, or NPs were placed in 900 mL of PBS (Phosphate Buffer Solution) 7.4 medium at

Nanoparticles were easily formed via ionic gelation due to the interactions between positive

Optimization of particle size was carried out using response surface methodology and central composite design of experiments. In previous experiments, several characteristics of the process were studied. Nanoparticles were formed using chitosan of high, medium, and low

*D* = (2)

h

etry using a Zetamaster analyzer system (Malvern Instruments, UK).

and sodium alginate were added directly before the compression process.

**Table 1.** Experiments corresponding to the central composite design study.

Analysis of variance (ANOVA) for particle size and polydispersity index allows us to identify which factors and interactions between factors influence the responses. Pareto chart indicates the statistically significant factors in order to build the response surface mathematical model (**Figure 4**). Subsequently, the mathematical model can be built to predict the response depending on the value taken by the factors.

**Figure 4.** Pareto chart (Factor A = chitosan concentration, Factor B = complex concentration, and Factor C = sodium sulfate concentration).

The model obtained for the optimization of particle diameter explains 98.3% of the variability in the experimental data, whereas for the optimization of polydispersity index, the model obtained explains only 88% of the variability of experimental data. **Figure 5** shows a schematic representation of the response surface for a pair of factors influencing the particle size.

**Figure 5.** Schematic representation of response surface for particle diameter as a function of DF-HPβCD and sodium sulfate concentration (variation in the third factor cannot be shown in the same figure; for this reason, chitosan concentration has been set on 0.3%).

The three-dimensional representation of the response as a function of two factors (**Figure 4**) does not allow us to visualize the minimum value for the selected surface. **Figure 6** allows us to carry out a preliminary analysis of the influence of each individual factor and its interactions in the response.

(**Figure 4**). Subsequently, the mathematical model can be built to predict the response depend-

**Figure 4.** Pareto chart (Factor A = chitosan concentration, Factor B = complex concentration, and Factor C = sodium

The model obtained for the optimization of particle diameter explains 98.3% of the variability in the experimental data, whereas for the optimization of polydispersity index, the model obtained explains only 88% of the variability of experimental data. **Figure 5** shows a schematic representation of the response surface for a pair of factors influencing the particle size.

**Figure 5.** Schematic representation of response surface for particle diameter as a function of DF-HPβCD and sodium sulfate concentration (variation in the third factor cannot be shown in the same figure; for this reason, chitosan concen-

The three-dimensional representation of the response as a function of two factors (**Figure 4**) does not allow us to visualize the minimum value for the selected surface. **Figure 6** allows us to carry out a preliminary analysis of the influence of each individual factor and its interactions

ing on the value taken by the factors.

114 Biological Activities and Application of Marine Polysaccharides

sulfate concentration).

tration has been set on 0.3%).

in the response.

**Figure 6.** Schematic representation of the influence of each factor and the interactions between factors in the particle diameter (Factor A = chitosan concentration, Factor B = complex concentration, and Factor C = sodium sulfate concentration).

The minimum particle diameter was obtained for concentrations of chitosan 0.12%, DF-HPβCD 0.34%, and sodium sulfate 2.2% according to the prediction of the mathematical model. Moreover, the minimum polydispersity index was obtained for concentrations of chitosan 0.61% and sodium sulfate 1.2% (DF-HPβCD concentration is not an influence factor for PDI).

**Figure 7.** Schematic representation of response surface for convenience of responses as a function of DF-HPβCD and sodium sulfate concentration (variation in the third factor cannot be shown in the same figure; for this reason, chitosan concentration has been set on 0.3%).

As can be observed, the optimization of both responses leads to different values for the three factors. It is therefore necessary to define a new function that optimizes the convenience of both responses simultaneously. For that purpose, a weight three times greater was conferred for the particle diameter than for polydispersity index. The representation of the response surface desirability function is shown in **Figure 7**. The values of factors for obtaining the best nanoparticles in terms of particle diameter and polydispersity index simultaneously are chitosan 0.14%, DF-HPβCD 0.28%, and sodium sulfate 1.9%.

Once the model that describes the function convenience was built, the nanoparticles were performed using the optimal conditions calculated showing a particle diameter of 515 nm and a polydispersity index of 0.14. The optimized nanoparticles showed a formation yield of 72.6%, an encapsulation efficiency of 34% and the amount of drug encapsulated was 42.3 μg/mg.

#### **3.2. Characterization of nanoparticles**

After the optimization from based on response surface methodology, physicochemical characterization of the optimized formulation was carried out.

It is important to ensure that all drug molecules are contained inside the nanoparticle and not distributed on the surface in order to prevent releases in two stages when it is intended to obtain controlled release systems [27]. With the aim to verify the distribution of drug in these systems, X-ray powder diffractometry was carried out (**Figure 8**). The absence of drug crystals on nanoparticle surface was corroborated, so a release in one single mode could be expected.

**Figure 8.** X-ray diffraction patterns of diflunisal (―), chitosan empty nanoparticles ( ), and optimized nanoparticle formulation CS-(DF-HPβCD) ( ).

The study of nanoparticle morphology was carried out by transmission electron microscopy. Round shape of nanoparticles can be visualized in **Figure 9**, confirming the previously measured particle size by dynamic light scattering (DLS), which reported a particle diameter distribution between 500 and 550 nm. The size measured by DLS is slightly higher than shown in the TEM images. This fact is due to the measurements of hydrodynamic radius obtained by DLS (which include solvent molecules displaced together with solid nanoparticles) and is always higher than the real size of nanoparticles. The zeta potential of the systems was 33.2 mV ensuring the stability of the nanoparticles in solution.

**Figure 9.** TEM image of the optimized nanoparticle formulation CS-(DF-HPβCD).

#### **3.3. Characterization of solid dispersion systems**

a polydispersity index of 0.14. The optimized nanoparticles showed a formation yield of 72.6%, an encapsulation efficiency of 34% and the amount of drug encapsulated was 42.3 μg/mg.

After the optimization from based on response surface methodology, physicochemical

It is important to ensure that all drug molecules are contained inside the nanoparticle and not distributed on the surface in order to prevent releases in two stages when it is intended to obtain controlled release systems [27]. With the aim to verify the distribution of drug in these systems, X-ray powder diffractometry was carried out (**Figure 8**). The absence of drug crystals on nanoparticle surface was corroborated, so a release in one single mode could be expected.

**Figure 8.** X-ray diffraction patterns of diflunisal (―), chitosan empty nanoparticles ( ), and optimized nanoparticle

The study of nanoparticle morphology was carried out by transmission electron microscopy. Round shape of nanoparticles can be visualized in **Figure 9**, confirming the previously measured particle size by dynamic light scattering (DLS), which reported a particle diameter distribution between 500 and 550 nm. The size measured by DLS is slightly higher than shown in the TEM images. This fact is due to the measurements of hydrodynamic radius obtained by DLS (which include solvent molecules displaced together with solid nanoparticles) and is always higher than the real size of nanoparticles. The zeta potential of the systems was 33.2

**3.2. Characterization of nanoparticles**

116 Biological Activities and Application of Marine Polysaccharides

formulation CS-(DF-HPβCD) ( ).

mV ensuring the stability of the nanoparticles in solution.

characterization of the optimized formulation was carried out.

Diflunisal and chitosan solid dispersions were analyzed by X-ray diffractometry (XRD) and scanning electron microscopy (SEM). It is important to characterize the crystalline state of drug molecules if it will be administered by oral route. Drugs in crystalline state usually show a lower dissolution rates, because it is necessary to use a certain amount of energy to break the crystal lattice formed by drug molecules.

**Figure 10.** XRD patterns of diflunisal (–), DF-CS kneaded system ( ), and chitosan ( ).

The X-ray diffraction patterns of pure components, diflunisal and chitosan, and DF-CSkneaded systems are shown in **Figure 10**. Chitosan shows the typical profile of amorphous materials. Diflunisal diffraction pattern showed the peaks of polymorph II [28, 29]. The kneading of diflunisal and chitosan gave rise to amorphous systems, and the X-ray diffraction pattern showed the absence of reflections as a consequence of the breaking of crystal lattice formed by molecules of diflunisal, which is molecularly dispersed within the amorphous polymer.

SEM micrographs show diflunisal as long prismatic, rodlike crystals typical of crystalline form II [19]. Chitosan particles showed rough surface with very different particle sizes. The disappearance of diflunisal needle agglomerates when the complex was observed corroborates the interaction between drug and polymer (**Figure 11**).

**Figure 11.** SEM micrographs of chitosan (A), diflunisal (B), and DF-CS-kneaded system (C).

#### **3.4. Dissolution rate studies**

#### *3.4.1. Dissolution from tablets*

Drug solubility enhancement caused by interaction with chitosan has been described previously in the literature [19]. The interaction by hydrogen bonds between diflunisal and chitosan is the main reason by which a faster release of diflunisal can be expected when it is compressed in the presence of chitosan. Different agents commonly used in the pharmaceutical industry have been employed to modulate diflunisal release rate from solid dispersions formed with chitosan by kneading. The dissolution rates from the different matrices prepared are shown in **Figure 12**.

Diflunisal release from tablets formed by kneading with chitosan showed a very fast release of diflunisal; in this case, the maximum amount of diflunisal was released to the medium in less than 1 h. In order to modulate drug release rate, it is therefore necessary to employ agents able to delay drug delivery. Sodium alginate and hydroxypropyl methylcellulose are widely used in the pharmaceutical domain for this purpose. These agents cause matrix gelification in aqueous solution promoting the slowly release of drug by diffusion from inside the matrix. For both concentrations of alginate, the plateau was reached at 15 h; in case of the addition of HPMC, the complete drug release was not achieved until 30 h, thereby achieving a much slower release.

pattern showed the absence of reflections as a consequence of the breaking of crystal lattice formed by molecules of diflunisal, which is molecularly dispersed within the amorphous

SEM micrographs show diflunisal as long prismatic, rodlike crystals typical of crystalline form II [19]. Chitosan particles showed rough surface with very different particle sizes. The disappearance of diflunisal needle agglomerates when the complex was observed corroborates

the interaction between drug and polymer (**Figure 11**).

118 Biological Activities and Application of Marine Polysaccharides

**Figure 11.** SEM micrographs of chitosan (A), diflunisal (B), and DF-CS-kneaded system (C).

Drug solubility enhancement caused by interaction with chitosan has been described previously in the literature [19]. The interaction by hydrogen bonds between diflunisal and chitosan is the main reason by which a faster release of diflunisal can be expected when it is compressed in the presence of chitosan. Different agents commonly used in the pharmaceutical industry have been employed to modulate diflunisal release rate from solid dispersions formed with chitosan by kneading. The dissolution rates from the different matrices prepared are shown

Diflunisal release from tablets formed by kneading with chitosan showed a very fast release of diflunisal; in this case, the maximum amount of diflunisal was released to the medium in less than 1 h. In order to modulate drug release rate, it is therefore necessary to employ agents able to delay drug delivery. Sodium alginate and hydroxypropyl methylcellulose are widely used in the pharmaceutical domain for this purpose. These agents cause matrix gelification in aqueous solution promoting the slowly release of drug by diffusion from inside the matrix. For both concentrations of alginate, the plateau was reached at 15 h; in case of the addition of HPMC, the complete drug release was not achieved until 30 h, thereby achieving a much slower

**3.4. Dissolution rate studies**

*3.4.1. Dissolution from tablets*

in **Figure 12**.

release.

polymer.

**Figure 12.** Dissolution rate of diflunisal from tablets compressed DF-CS systems alone (■) or in combination with 20% alginate ( ), 40% alginate ( ), and 40% HPMC ( ). Tablets compressed with pure diflunisal ( ).

Kinetic parameters were calculated with the aim to facilitate a rapid and easy comparison of release profiles. Dissolution efficiency (DE) is defined as the percentage of area under the dissolution curve at a fixed time. It is expressed as a percentage of the rectangular area described by 100% dissolution in the same time [30]:

$$\begin{aligned} \stackrel{\circ}{}\_{DE} &= \frac{\int \nu \times dt}{\nu\_{100} \times t} \times 100 \end{aligned} \tag{3}$$

$$MDT = \frac{\sum\_{j=1}^{n} \hat{t}\_j \times \Delta M\_j}{\sum\_{j=1}^{n} M\_j} \tag{4}$$

For calculating mean dissolution time (MDT), *j* is the sample number, is the time at the midpoint between *tj* and *tj*−1, and Δ*Mj* is the additional amount of drug dissolved between *tj* and *tj*−1. *M*12h indicates the amount of drug released at a fixed time (12 h). *t*5% indicates the time needed to release a fixed amount of drug (5%). The trends exhibited by different profiles according to the agents used can be quantified from the kinetic parameters shown in **Table 2**.

**Table 2.** Dissolution parameters for diflunisal release from different tablets.

As can be observed, DE value closes to 100% and low mean dissolution time indicates a fast release of diflunisal from chitosan matrix causing this release that could be considered as bursttype profile. When alginate or HPMC was added to DF-CS system, a significant decrease in the dissolution rate was found, being more effective in case of HPMC as was evidenced by the low DE value and highs MDT and *t*5% values.

To test whether the addition of a higher amount of alginate results in a significant change in the rate of drug release, the comparison between different profiles can also be performed by pair-wise procedures, including difference and similarity factors (**Table 3**). The different factor (*f*1) measures the percent error between two curves over all time points, where *n* is the number of sampling points and *Rj* and *Tj* are the percent dissolved of the reference and test products at each time point *j.* Similar profiles yield *f*1 values lower than 15. Similarity factors (*f*2) higher than 50 indicate similar release profiles. These parameters can be calculated by the following equations:

$$\sum\_{j=1 \atop j=1}^{n} \left| R\_j - T\_j \right| \tag{5}$$

$$\sum\_{j=1}^{n} R\_j \tag{5}$$

$$\|f\_2 = 50 \times \log \left\{ \left[ 1 + \left( \frac{1}{n} \right) \sum\_{j=1}^{n} \left| R\_j - T\_j \right|^2 \right]^{-4/2} \times 100 \right\} \tag{6}$$

**Table 3.** Comparative parameters of diflunisal release profiles.

From the data obtained for difference and similarity factors, it can be concluded that the addition of HPMC or alginate leads to different dissolution rates compared to DF-CS systems. Different amounts of alginate (20% vs 40%) do not involve significantly changes in diflunisal release (*f*1 < 15 and *f*2 > 50). However, the addition of alginate and HPMC clearly proves to be significant in the dissolution rate of the drug. The different dissolution rate reduction between alginate and HPMC is also evident by comparison through *f*1 and *f*2.

**Formulation tablets DE (%) MDT (h)** *M***12h (mg/mL)** *t***5% (h)** DF-CS 94,2 0.17 64.90 <0.3 DF-CS-Alg (20%) 68.8 2.03 60.85 0.3 DF-CS-Alg (40%) 60.4 2.96 58.41 0.3 DF-CS-HPMC (40%) 31.7 8.83 34.16 0.7

As can be observed, DE value closes to 100% and low mean dissolution time indicates a fast release of diflunisal from chitosan matrix causing this release that could be considered as bursttype profile. When alginate or HPMC was added to DF-CS system, a significant decrease in the dissolution rate was found, being more effective in case of HPMC as was evidenced by the

To test whether the addition of a higher amount of alginate results in a significant change in the rate of drug release, the comparison between different profiles can also be performed by pair-wise procedures, including difference and similarity factors (**Table 3**). The different factor (*f*1) measures the percent error between two curves over all time points, where *n* is the

products at each time point *j.* Similar profiles yield *f*1 values lower than 15. Similarity factors (*f*2) higher than 50 indicate similar release profiles. These parameters can be calculated by the

100

*j j*

1 2 2

are the percent dissolved of the reference and test

å (6)

(5)

and *Tj*

1

=

*j n j j*

*f R T n*

*n*

å

1

=

å

*j j*

*R T*

*R*

1 1 50 log 1 100 *n*

**Profiles compared** *f***1** *f***2** DF-CS vs DF-CS-Alg (20%) 71 26 DF-CS-Alg (20%) vs DF-CS-Alg (40%) 13 62 DF-CS-Alg (40%) vs DF-CS-HPMC (40%) 78 19

From the data obtained for difference and similarity factors, it can be concluded that the addition of HPMC or alginate leads to different dissolution rates compared to DF-CS systems.

= ì ü é ù ï ï æ ö =´ + - ´ í ý ê ú ç ÷ ï ï è ø ë û î þ

*j*

1

*f*

**Table 2.** Dissolution parameters for diflunisal release from different tablets.

low DE value and highs MDT and *t*5% values.

120 Biological Activities and Application of Marine Polysaccharides

2

**Table 3.** Comparative parameters of diflunisal release profiles.

number of sampling points and *Rj*

following equations:

In the light of the different dissolution profiles shown in **Figure 12**, the study of the mechanisms involved in drug release from the different matrices is important for a better understanding of the phenomena involved in this process.

The *Korsmeyer-Peppas* equation is a simple semiempirical model which relates drug release with the elapsed time [31]. *n* Values indicate release exponent; for our systems, when it is close to 0.43 means that drug release occurs by pure Fickian diffusion process, when relaxation phenomena and erosion of matrix are involved in drug release, the *n*-value is approaching to 1. *kKP* is a constant incorporating the structural and geometrical characteristics of the matrix system:

$$\frac{M\_{\ll}}{M\_{\ll}} = k\_{\ll} \mathfrak{r}^{\prime} \tag{7}$$

The *Peppas-Sahlin* model fitted to the experimental data provides values of both diffusion constant (*kD*) for the Fickian contribution and erosion constant (*kE*) for the erosional/relaxational contribution, the *m* exponent is 0.43 for our systems [32]:

$$\frac{M\_{\rm \cdot}}{M\_{\rm \cdot \cdot}} = k\_D t^m + k\_E t^{2m} \tag{8}$$

Kinetic fitting to aforementioned mathematical models was carried out in order to analyze the phenomena involving in drug release (**Table 4**).

**Table 4.** Kinetic constants and correlation coefficients after data fitting to Korsmeyer-Peppas and Peppas-Sahlin equations (DF-CS systems could not be fitted to kinetic equations because of the fast drug release rate corresponding to burst type).

As can be observed in the values shown in **Table 4**, a mixture of phenomena that influence drug release to the dissolution medium occurs for the systems studied (except DF-CS). In order to assess the contribution of erosion and diffusion phenomena, erosion (E) and diffusion (D) relative contributions have been calculated [33] and represented as function of time in **Figure 13**:

$$D = \frac{1}{1 + \left(\bigvee\_{k} k\_{E}\right)t^{m}}\tag{9}$$

$$1 = D + E \tag{10}$$

**Figure 13.** Erosion (empty points) and diffusion (filled points) relative contribution for DF-CS-Alg (20%) (A), DF-CS-Alg (40%) (B), and DF-CS-HPMC (40%) (C).

In all systems, the erosion contribution begins as less important than the diffusion contribution. However, this trend was inverted for the three systems. For each system a time in which the erosion is becoming more important than diffusion can be calculated. For Alg-20% system, this change occurs around 12 h while occurring at 6 h for Alg-40% system and in less than 2 h for HPMC-40% system. This contribution ratio explains why drug release from system containing HPMC is slower; in fact, the amount of drug release is practically independent of time since the erosion phenomenon reaches a greater importance than the contribution of diffusion phenomena. The same trend, but at different time, can be observed for alginate systems where the diffusion phenomenon remains longer.

#### *3.4.2. Dissolution from nanoparticles*

Diflunisal release from the previously optimized nanoparticle formulation has been studied. The same amount of diflunisal contained in nanoparticles was tested for comparison purposes. Drug dissolution profiles from nanoparticle system and pure diflunisal are shown in **Figure 14**.

**Figure 14.** Dissolution rate of diflunisal from diflunisal powder ( ) and CS-(DF-HPβCD) nanoparticles ( ).

Diflunisal release from nanoparticles has proven to be faster than directly from diflunisal powder. The total amount of drug encapsulated into nanoparticles was released within 6 h. However, the amount of diflunisal released in the same time is less than 10 mg/L when drug release occurs from diflunisal powder directly. This fact is due on one hand to the increase in drug solubility produced by the presence of small amounts of chitosan and HPβCD in solution; on the other hand, diflunisal molecules in nanoparticles are presented in the amorphous state, which allow to a faster release than in the case of pure diflunisal, where an additional energy is needed to break the crystal lattice.

The dissolution efficiency was 69.5% for nanoparticle system and 18.2% for pure diflunisal. This difference shows clearly a faster dissolution rate and a greater amount of drug released.

Mathematical models cannot be applied to these systems, which do not have a controlled and constant geometry that allows ascertain what proportion involved diffusion and erosion/ relaxation phenomena.

## **4. Conclusions**

to assess the contribution of erosion and diffusion phenomena, erosion (E) and diffusion (D) relative contributions have been calculated [33] and represented as function of time in

> 1 1 *E m D*

*k t k* = æ ö + ç ÷

**Figure 13.** Erosion (empty points) and diffusion (filled points) relative contribution for DF-CS-Alg (20%) (A), DF-CS-

In all systems, the erosion contribution begins as less important than the diffusion contribution. However, this trend was inverted for the three systems. For each system a time in which the erosion is becoming more important than diffusion can be calculated. For Alg-20% system, this change occurs around 12 h while occurring at 6 h for Alg-40% system and in less than 2 h for HPMC-40% system. This contribution ratio explains why drug release from system containing HPMC is slower; in fact, the amount of drug release is practically independent of time since the erosion phenomenon reaches a greater importance than the contribution of diffusion phenomena. The same trend, but at different time, can be observed for alginate systems where

Diflunisal release from the previously optimized nanoparticle formulation has been studied. The same amount of diflunisal contained in nanoparticles was tested for comparison purposes. Drug dissolution profiles from nanoparticle system and pure diflunisal are shown in **Figure 14**.

è ø (9)

1 = + *D E* (10)

*D*

122 Biological Activities and Application of Marine Polysaccharides

**Figure 13**:

Alg (40%) (B), and DF-CS-HPMC (40%) (C).

the diffusion phenomenon remains longer.

*3.4.2. Dissolution from nanoparticles*

Chitosan can interact with a high variety of drugs due to its physicochemical properties. The abundance and low cost associated with the production of chitosan make that it can be considered as a promising material in pharmaceutical industry. Chitosan is a versatile product able to be chemically modified and the ease to control the length of its polymer chains makes chitosan an ideal product for use in controlled release formulations.

Response surface methodology is a good tool to optimize fabrication process of pharmaceutical formulations when it depends on two or more factors. It is a good strategy to minimize the number of experiments and to avoid mistakes usually made when the factors involved in the process are optimized individually.

The use of chitosan alone or in the presence of other excipients is made possible to obtain delivery systems on demand in solid oral dosage forms as tablets or nanoparticles. It allows us to modulate drug release from close to zero-order to type-burst kinetics.

## **Acknowledgements**

The authors thank the University of Navarra (PIUNA project) for the financial support and the ADA for the grant of David Lucio.

#### **Abbreviations**

## **Author details**

David Lucio\* and María Cristina Martínez-Ohárriz

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Chemistry, Faculty of Sciences, University of Navarra, Navarra, Spain

## **References**

number of experiments and to avoid mistakes usually made when the factors involved in the

The use of chitosan alone or in the presence of other excipients is made possible to obtain delivery systems on demand in solid oral dosage forms as tablets or nanoparticles. It allows

The authors thank the University of Navarra (PIUNA project) for the financial support and the

us to modulate drug release from close to zero-order to type-burst kinetics.

process are optimized individually.

124 Biological Activities and Application of Marine Polysaccharides

**Acknowledgements**

CS Chitosan DF Diflunisal

SS Sodium sulfate Alg Sodium alginate

NPs Nanoparticles

**Abbreviations**

ADA for the grant of David Lucio.

HPβCD 2-Hydroxypropyl-β-cyclodextrin

HPMC Hydroxypropyl methylcellulose

RSM Response surface methodology CCD Central composite design DTA Differential thermal analysis SEM Scanning electron microscopy TEM Transmission electron microscopy

DLS Dynamic light scattering PDI Polydispersity index

and María Cristina Martínez-Ohárriz

Department of Chemistry, Faculty of Sciences, University of Navarra, Navarra, Spain

\*Address all correspondence to<EMAIL_ADDRESS>
**Author details**

David Lucio\*

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## **Chapter 7**

**Provisional chapter**

## **Marine Polysaccharides as Multifunctional Pharmaceutical Excipients Marine Polysaccharides as Multifunctional Pharmaceutical Excipients**

G. Thirumurugan and M.D. Dhanaraju G. Thirumurugan and M.D. Dhanaraju

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66191

#### **Abstract**

This chapter is presented to depict the chance of marine polysaccharides satisfying the properties of good pharmaceutical excipients and the potential of that could be utilized as multifunctional pharmaceutical excipients in the pharmaceutical solid dosage form manufacturing as fillers, diluents/vehicles, binders, glidants, binders and disintegrants, and so on. In addition, this chapter discusses the use of marine polysaccharide holding a specific pharmacological activity in the formulation/dosage form, which is used for the same aliment/disease.

**Keywords:** marine polysaccharide, pharmaceutical excipients, bioavailability, drug delivery, dissolution, disintegrants, pharmacological activity

## **1. Introduction**

Drugs, which are obtained from plants, animals, marine source or minerals, are not very often administered in their pure form. Before releasing into the market, these drugs are combined with a variety of inert substances (excipients/adjuvants) and modified into a dosage form, which is convenient to be administered by a specified route [1]. Similar to the drugs, these excipients are also obtained from many sources, namely natural, synthetic sources. Nowadays, the naturally obtained excipients have shown more interest due to the various advantages like low cost, biocompatibility, biodegradability, non-toxicity, easy availability, eco-friendly processing, superior patient tolerance and acceptance [2]. Before, it was thought that the therapeutic reaction to a drug is a characteristic of its inherent pharmacological action. However, nowadays, it is much implicit that the dose-response connection obtained after drug administration by various routes—for example, oral and parenteral—is not identical. The difference is also observed when the same medicine is administered as dissimilar dosage forms or similar

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

formulations produced by dissimilar manufacturers, which in turn depends upon the physical, chemical properties of the drug, the excipients present in the formulation, the technique of formulation and the way of administration [1].

Natural marine origins are among the most widely used excipients in the formulation of different dosage forms. A number of marine-based polysaccharides, such as agar, alginate, carrageenan, fucoidan, chitosan and hyaluronan are utilized in pharmaceutical dosage form as binders, vehicles, disintegrating agents, gelling agents and drug release sustaining agents [3].

The purpose of this review is to discuss potential pharmaceutical formulation development applications of marine polysaccharides, with a special emphasis in multifunctional excipients development for enhancing bioavailability, drug delivery applications. In addition, this review discusses the use of marine polysaccharide holding a specific pharmacological activity in the formulation/dosage form, which is used for the same aliment/disease, for example, incorporating the marine polysaccharide having anti-cancer activity in anti-cancer drug formulation as excipients. In fact, data has added that such excipients operate on well-distinct biological pathways and receptors in order to implement their valuable properties. If additives that work on several cancer pharmacological pathways or receptors targets are jointed together in concentrations that are at and/or more than their recommended levels, then there is a possibility exists that such a formulated dosage form may offer "standalone" control of cancer.

## **2. Marine polysaccharides, as multifunctional pharmaceutical excipients**

A drug injected intravascularly (I.V/I.A) directly enters the blood and produces its pharmacological effects. The majority of drugs are administered extravascularly, usually through oral route. If anticipated to act systemically, such drugs can produce their pharmacological actions only when they come into the blood circulation from their site of application. In order to reach the blood circulation, orally administered formulation must disintegrate, deaggregate and dissolution of the drug in the aqueous fluid at the absorption site must occur [4]. If the drugs are not hydrophilic in nature, the absorption process of drugs like these is usually dissolution rate-limited (**Figure 1**).

Better absorption can be achieved by altering the characteristics of the dosage form using pharmaceutical excipients. Moreover, pharmaceutical excipients help in the manufacturing process by serving as a binder, diluents, wetting agent, filling agent, disintegrating agents, dissolution enhancers, and so on. [3]. Now their increasing demands and expectations

**Figure 1.** The two rate-determining steps in the absorption of orally administered dosage form.

with regard to quality have stimulated the development of new additives characterized by advanced assay and lesser content of impurities. Quality of formulation does depend on the quality of excipient [4]. The purpose of this study is to give a scope for the researchers to try and develop a formulation that is made up of just the drug and natural marine polysaccharides. This review is done to depict the chance of some marine polysaccharides satisfying the properties of good pharmaceutical excipients if the stability of them has been explored sufficiently.

#### **2.1. As binder**

formulations produced by dissimilar manufacturers, which in turn depends upon the physical, chemical properties of the drug, the excipients present in the formulation, the technique

Natural marine origins are among the most widely used excipients in the formulation of different dosage forms. A number of marine-based polysaccharides, such as agar, alginate, carrageenan, fucoidan, chitosan and hyaluronan are utilized in pharmaceutical dosage form as binders, vehicles, disintegrating agents, gelling agents and drug release sustaining agents [3]. The purpose of this review is to discuss potential pharmaceutical formulation development applications of marine polysaccharides, with a special emphasis in multifunctional excipients development for enhancing bioavailability, drug delivery applications. In addition, this review discusses the use of marine polysaccharide holding a specific pharmacological activity in the formulation/dosage form, which is used for the same aliment/disease, for example, incorporating the marine polysaccharide having anti-cancer activity in anti-cancer drug formulation as excipients. In fact, data has added that such excipients operate on well-distinct biological pathways and receptors in order to implement their valuable properties. If additives that work on several cancer pharmacological pathways or receptors targets are jointed together in concentrations that are at and/or more than their recommended levels, then there is a possibility

exists that such a formulated dosage form may offer "standalone" control of cancer.

**2. Marine polysaccharides, as multifunctional pharmaceutical excipients**

A drug injected intravascularly (I.V/I.A) directly enters the blood and produces its pharmacological effects. The majority of drugs are administered extravascularly, usually through oral route. If anticipated to act systemically, such drugs can produce their pharmacological actions only when they come into the blood circulation from their site of application. In order to reach the blood circulation, orally administered formulation must disintegrate, deaggregate and dissolution of the drug in the aqueous fluid at the absorption site must occur [4]. If the drugs are not hydrophilic in nature, the absorption process of drugs like these is usually dissolution

Better absorption can be achieved by altering the characteristics of the dosage form using pharmaceutical excipients. Moreover, pharmaceutical excipients help in the manufacturing process by serving as a binder, diluents, wetting agent, filling agent, disintegrating agents, dissolution enhancers, and so on. [3]. Now their increasing demands and expectations

**Figure 1.** The two rate-determining steps in the absorption of orally administered dosage form.

of formulation and the way of administration [1].

130 Biological Activities and Application of Marine Polysaccharides

rate-limited (**Figure 1**).

Marine polysaccharides can serve as a good binder in the production of tablets by the wet granulation method of manufacture. In this role, binders are either added as a solution or as a solid into the powder mix (following which the granulating fluid, typically water, is added). Due to the high concentration of hydroxyl groups in the polysaccharide, generally have a high water-binding capacity that makes wet granulation easier [5]. The responsible bio-adhesives marine polysaccharides have extraordinarily high cohesive strength and binding strength to the solid surfaces, enabling the API to remain attached under tensional conditions.

#### **2.2. As diluents**

Marine polysaccharides can be employed as diluents/fillers in the formulation of tablets (by all methods) to increase the mass of the solid dosage forms that hold a low concentration of therapeutic agent and thereby render the manufacturing process more reliable and reproducible [6]. For example, chitin and chitosan are used as a diluent or filler and as a binder in direct compression of tablet processing, as a disintegrant, and so on. Chitin and chitosan have a lowest bulk and tapped density that cause good flow as well as compaction during filling and tablet compression processing. **Figure 2** shows the structure of cellulose chitin and chitosan.

**Figure 2.** Structure of cellulose, chitin and chitosan.

**Figure 3.** Disintegrants facilitating the breakdown of the tablet into granules.

#### **2.3. As disintegrants**

Disintegrants are materials added to the dosage forms that enhance the breakup or disintegration of tablet formulations into smaller particles that dissolve faster than in the absence of disintegrants. These materials have the main role to oppose the efficiency of tablet binder and physical forces that behave under compression to form the tablets. Tablet disintegrant usually considered as the rate determining step (RDS) in a faster drug release. Marine polysaccharides may behave as a good disintegrating agent by increasing the porosity, wettability and wicking or capillary action and operate by swelling in the presence of aqueous fluids in tablet formulations to facilitate the breakdown of the tablet into granules upon entry into the stomach [7] (**Figure 3**).

#### **2.4. As dissolution enhancers**

Marine polysaccharides may serve as a dissolution enhancer for the poor soluble drug. These powders can reduce cohesive forces holding a tablet dosage form together and induce the breakup into smaller granules, thus increasing the effective surface area for dissolution (**Figure 4**).

**Figure 4.** Dissolution enhancer facilitating dissolution for the poor soluble drug.

#### **2.5. As drug delivery carriers**

**Figure 3.** Disintegrants facilitating the breakdown of the tablet into granules.

132 Biological Activities and Application of Marine Polysaccharides

Disintegrants are materials added to the dosage forms that enhance the breakup or disintegration of tablet formulations into smaller particles that dissolve faster than in the absence of disintegrants. These materials have the main role to oppose the efficiency of tablet binder and physical forces that behave under compression to form the tablets. Tablet disintegrant

**2.3. As disintegrants**

Marine polysaccharides have been widely used to synthesize drug delivery carriers. They are bio-compatible, non-toxic and bio-degradable and stimuli-responsive makes marine polysaccharides appropriate sources for the building of complex loading devices with a release that can be effectively controlled [4]. **Table 1** shows the compilation of marine polysaccharides utilized as drug delivery carriers, and **Figure 5** illustrates the mechanisms of various marine polysaccharide carriers and its method of preparation.

Drug delivery devices can be constructed using various methods and can be synthesized in a variety of shapes, such as hydro gels, micro or nanoparticles and capsules, capable of protecting a variety of bioactive agents such as proteins and nucleic acids.

**S. no**

1.

2. 3.

4.

Ulvans

Green algae

Cosmetic delivery [22], nano-fibres (tissue engineering and regenerative

medicine) [23], hydrogel [24]

 Fucoidans

Brown algae

Microspores, fucospheres [19], insulin controlled release [20],

nano-particles [21]

Carrageenans

Red algae

Tablet compressor [15], controlled release (temperature sensitive) [16], |fast

134 Biological Activities and Application of Marine Polysaccharides

release [17], sustained release [18] (Ph sensitive)

 Alginate

**Name of marine polysaccharide**

**Source** Brown sea weed

**Drug delivery use**

particles [13, 14]

Excipient, stabilizer [8], hydrogel matrices, beads, particles [9–12], micro

**Figure 5.** Illustrating the mechanism of various marine polysaccharide carriers and their method of preparation.

## **3. Pharmacological activity of marine polysaccharides and potential possibilities of standalone effects when incorporating as excipients**

Marine polysaccharides stand for a number of abundant bio-active substances in marine organisms. In fact, numerous marine macro- and microorganisms are good quality possessions of carbohydrates with miscellaneous applications due to their bio-functional efficacies. By involving on cell propagation and cycle, and by modulating metabolic pathways, marine polysaccharides have numerous pharmacological efficacies, such as antioxidative, antibacterial, antiviral, immuno-stimulatory, anticoagulant and anticancer effects. Besides the polysaccharides, monosaccharides are useful for humans and can cure many diseases, mainly those linked to metabolism deficiency such as diabetes.

There has been growing facts in recent years that pharmaceutical additives may not be harmless, "inert" components of a formulation, but may hold either "stand alone" pharmacological activity or may act to modify the pharmacological efficacy of the API. Because most excipients have historically derived from food or food products, they have been assigned—and in most cases, experimentally confirmed—to have widespread pharmacological activity based on the ingestion of those products. Marine polysaccharides holding a specific pharmacological activity can be incorporated as an excipient in the formulation/dosage form, which is used for the same aliment/disease. **Table 2** shows the pharmacological activity of marine polysaccharides, and **Figure 6** illustrates the mechanism of action involved in the pharmacological activity of marine polysaccharides.

**S. no**

9.

10.

11.

 Agarose

Red algae **Table 1.** Compilation of marine polysaccharides utilized as drug delivery carriers.

Hydrogels [36]

Keratan sulfate

Ray skin (glycosaminoglycans)

136 Biological Activities and Application of Marine Polysaccharides

Heparan sulfate

**Name of marine polysaccharide**

**Source** Ray skin (glycosaminoglycans)

Cancer treatment [35]

**Drug delivery use**

**Table 2.** Pharmacological activity of marine polysaccharides.

**Figure 6.** Illustrating the mechanism of action involved in the pharmacological activity of marine polysaccharides.

However, the majority of the pathways or modes of action has not been revealed in humans. In addition, fairly only some have been investigated in-vitro. These in-vitro findings are unable to extrapolate into their actions in-vivo in humans. There should be well-controlled human clinical investigations using such a mixture of additives are essential prior to any rationalization for effectiveness can be asserted.

Ideation of using "marine polysaccharide bio-active excipients" is possible for diseases that are chronic in nature and for whom gradual and lasting beneficial efficacy may be observed from the modulation of signalling pathways that direct to incremental but visible improvements in the quality of life. Such "marine polysaccharide bio-active excipients" may not be efficient for acute diseases for which radical interventions are necessary.

## **4. Conclusion**

Marine polysaccharides have a huge potential as multifunctional pharmaceutical excipients for commercial needs. However, there is a need to characterize, check compatibility of these polysaccharides to produce pharmaceutical grade additives that could find the utility in drug dosage form. In addition, research is warranted in pharmacodynamic interaction of these marine polysaccharides and particular drug when using for same aliments.

## **Author details**

G. Thirumurugan\* and M.D. Dhanaraju

\*Address all correspondence to<EMAIL_ADDRESS>
Research Lab, GIET School of Pharmacy, Rajahmundry, Andhra Pradesh, India

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**Figure 6.** Illustrating the mechanism of action involved in the pharmacological activity of marine polysaccharides.

**S. no Marine polysaccharide Source Pharmacological activity**

(glycosaminoglycans)

(glycosaminoglycans)

(glycosaminoglycans)

(glycosaminoglycans)

and shark)

2. Carrageenans Red algae Anticoagulant [37], Anti-tumour [38] , Immuno-modulator

4. Ulvans Green algae Anti-viral, anti-oxidant, anti-coagulant, anti-tumour,

5. Chitosans Marine animal (chitin) Antimicrobial [49], anti-tumour and inflammatory [50]

11. Agarose Red algae Glucose intolerance in type 2 diabetes mellitus, weight loss,

[43] 3. Fucoidans Brown algae Anti-tumour [44, 45], anti-thrombin [19], anti-coagulant,

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Anti-coagulant [54]

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Anti-coagulant [53], supplement for arthritis [53]

1. Alginate Brown sea weed

138 Biological Activities and Application of Marine Polysaccharides

6. Hyaluronans Marine animal

8. Dermatan sulfate Ray skin

9. Heparan sulfate Ray skin

10. Keratan sulfate Ray skin

7. Chondroitin sulfate Marine animals (whale

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