**16. Safe biomaterial**

12 The Complex World of Polysaccharides

quality will also influence its biocompatibility [69.70].

responses to biomaterial implants [76-81].

**15. Biodegradability** 

the physical and chemical properties of the chitosan in order to improve its medicinal

The excellent biological properties of chitosan can be potentially improved with a variety of additional chemicals such as polyethylene glycol and carboxymethyl N-acyl groups in order to produce biocompatible chitosan derivatives for use as wound dressings [72]. Chitosan's positive surface charge enables it to effectively support cell growth [73]. Chitosan-gelatin sponge wound dressing had demonstrated a superior antibacterial effect. Additionally, chitosan gelatin sponge allowed the wound site to contract markedly and shortened the wound healing time, as compared with sterile Vaseline gauze [74]. Widely used surface modification techniques include coating, oxidation by low temperature plasma for better printing and adhesion and surfactant addition for antistatic. Blends are often used to improve tensile properties and to provide a stronger structural component for separation media that supports the active polymer. The physical properties of a polymer can also be altered by introducing a second polymer that improves the properties of the original polymer in certain aspects, such as hydrophobility, lowered melt temperature, raised glass transition temperature, etc [75]. A thorough understanding of cell and proteins interactions with artificial surfaces is of importance to design suitable implant surfaces and substrates. The surface properties of newly synthesized biomedical grade chitosan derivatives, including surface composition, wettability, domain composition, surface oxidation, surface charge and morphology, may influence protein adsorption and subsequently, the cellular

The claim "biodegradable" is often associated with environmentally friendly products. It is defined as being able to be broken down by natural processes, into more basic components.

An important aspect in the use of polymers as drug delivery systems is their metabolic fate in the body or biodegradation. In the case of the systemic absorption of hydrophilic polymers such as chitosan, they should have a suitable Mw for renal clearance. If the administered polymer's size is larger than this, then the polymer should undergo degradation. Biodegradation (chemical or enzymatic) provides fragments suitable for renal clearance. Chemical degradation in this case refers to acid catalysed degradation i.e. in the stomach. Enzymatically, chitosan can be degraded by enzymes able to hydrolyse glucosamine–glucosamine, glucosamine–N- acetyl-glucosamine and N-acetyl-glucosamine– N-acetyl- glucosamine linkages [83]. Even though depolymerisation through oxidation– reduction reaction [84] and free radical degradation [85] of chitosan have been reported

Chitosan is thought to be degraded in vertebrates predominantly by lysozyme and by bacterial enzymes in the colon [83, 86]. However, eight human chitinases (in the glycoside hydrolase 18 family) have been identified, three of which have shown enzymatic activity

Products are usually broken down by bacteria, fungi or other simple organisms [82].

these are unlikely to play a significant role in the in vivo degradation.

Chitosan is a potentially biologically compatible material that is chemically versatile (–NH2 groups and various Mw). These two basic properties have been used by drug delivery and tissue engineering to create a great amount of formulations and scaffolds that show promise in healthcare. It is approved for dietary applications in Japan, Italy and Finland [91] and it has been approved by the FDA for use in wound dressings [92] but is not approved for any product in drug delivery. The term "Chitosan" represents a large group of structurally different chemical entities that may show different biodistribution, biodegradation and toxicological profiles.

The formulation of chitosan with a drug may alter the pharmacokinetic and biodistribution profile.The balancing, or reduction, of the positive charges on the chitosan molecule has effects on its interaction with cells and the microenvironment, often leading to decreased uptake and a decrease in toxicity. The modifications made to chitosan could make it more or less toxic and any residual reactants should be carefully removed. In addition, the route of administration determines the uptake, concentration, contact time and cell types affected.



**Table 1.** Toxicity of chitosan and chitosan derivatives Table taken from [94]

14 The Complex World of Polysaccharides

95% DD, 18.7 kDa Steric acid conjugation micelle

95% DD, 18.7 kDa teric acid conjugation and

entrapment in micelle

97% DD, 65 kDa N-octyl-O-sulphate Invitro, primary rat

**Modification Assessment IC50** 

hepatocytes

None, glutamic acid salt 0.56±0.10, 0.48±0.07,

None, Lactic acid salt 0.38±0.13, 0.31±0.06,

None, hydrochloride salt 0.23±0.13, 0.22±0.06,

None, lactic acid salt In vitro B16F10 cells 2.00±0.18 mg/ml

None, glutamic acid salt In vitro B16F10 cells 2.47±0.14 mg/ml

None, lactic acid salt In vitro B16F10 cells 1.73±1.39 mg/ml

In vitro, MCF7 and COS7 cells, 6 h & 24 h

pH 6.2

None, aspartic acid salt In vitro, Caco-2 cells,

78% DD, <50 kDa None, lactic acid salt In vitro B16F10 cells 2.50 mg/ml

100% DD, 152 kDa Glycol chitosan In vitro B16F10 cells 2.47±0.15 mg/ml

None, hydrochloric acid

None, hydrochloric acid

chitosan, chloride salt

salt

salt

100% DD, 3–6 kDa 20, 44, 55% Trimethyl

100% DD, 3–6 kDa 94% Trimethyl chitosan,

100% DD, 3–6 kDa 94% Trimethyl chitosan,

100% DD, 100 kDa 36% Trimethyl chitosan, chloride sal

100% DD, 100 kDa 36% Trimethyl chitosan, chloride sal

chloride salt

chloride salt

In vitro ,A549 cells 369±27 μg/ml

In vitro ,A549 cells 234±9 μg/ml

>200 mg/ml

mg/ml

mg/ml

mg/ml

mg/ml

>10 mg/ml

In vitro B16F10 cells 2.24±0.16 mg/ml

In vitro B16F10 cells 0.21±0.04 mg/ml

In vitro, MCF7, 6 h 1.402±0.210 mg/ml

In vitro, COS7, 6 h 2.207±0.381 mg/ml

In vitro, MCF7, 6 h 0.823±0.324 mg/ml

In vitro, COS7, 6 h >10 mg/ml

0.67±0.24, 0.61±0.10, 0.65±0.20, 0.72±0.16

0.35±0.06, 0.46±0.06

0.34±0.04, 0.37±0.08

0.27±0.08, 0.23±0.08

**Chitosan details (DD, MW)** 

87% DD, 20, 45, 200, 460 kDa

87% DD, 20, 45, 200, 460 kDa

87% DD, 20, 45, 200, 460 kDa

87% DD, 20, 45, 200, 460 kDa

82% DD, 150–170

>80% DD, 60–90

77% DD, 180–230

85% DD, 60–90

81% DD, 100–130

kDa

kDa

kDa

kDa

kDa

In a series of articles are described the effects of chitosans with differing molecular weights and degree of deacetylation in vitro and in vivo. Toxicity was found to be degree of deacetylation and molecular weight dependent. At high DD the toxicity is related to the molecular weight and the concentration, at lower DD toxicity is less pronounced and less related to the molecular weight [93].

A summary of toxicities of chitosan and derivatives assessed through IC50 values is presented in the next table [94].

From this table it can be gathered that most chitosans (and derivatives) are not significantly toxic compared to a toxic polymer such as polyethylenimine [94].

It appears that the toxicity of chitosan is related to the charge density of the molecule, toxicity increases with increasing density. It appears that there is a threshold level below which there are too few contact points between a molecule and the cell components to produce a significantly toxic effect. This balance is between 40 and 60% DD, or degree of trimethylation, although any sufficiently small chitosan (<10 kDa) is not appreciably toxic. Modifications that do not increase the charge on the molecule seem to have little effect on the toxicity beyond that of the native molecule [94].
