Strategy to improve the characterization of chitosan for sustainable biomedical applications: SAR guided
7.1 Introduction
Chitin and Chitosan are important biopolymers consisting of(1→ 4)-2-amino-2-deoxy-β-D-glucan (GlcN) and (1→4)-2-acetamido-2-deoxy-β-D-glucan (GlcNAc) units. Due to their interesting properties they are taking center stage for many different appli-cations e.g. waste water treatment152 (coagulant, heavy-metal binding112, protein binding), food industry (diet substance153, cholesterol reducer163) biomedical (gene delivery164, medical dressing34, artificial tissue/skin165, prothesis coating166), agricul-ture167(animal food additive168, seed/leaf coating169), pharmaceutical (anti-adhesive
170, drug delivery171, wound-healing172) and cosmetic uses (gelation agent, plaque in-hibition157, moisturizers173, stabilizers173).
For any of these applications sustainable materials are required. In the light of chapter 19 of Agenda 21174the following principles of green chemistry175are of main relevance:
”Chemical products should be designed to preserve efficacy of function while reducing toxicity”(4th principle).
”A raw material or feedstock should be renewable rather than depleting whenever tech-nically and economically practicable” (7th principle).
”Chemical products should be designed so that at the end of their function they break-down into innocuous degradation products and do not persist in the environment”
(10th principle).
Chitosan has the potential to substitute conventional materials in many applications mentioned above. Typical synthetic materials, which are usually applied, may be toxic, not renewable and with unknown degradation pathway possibly resulting in (eco)toxic metabolites. In a consequence the possibility of these negative impacts demands for time and money consuming tests to ensure the safety of synthetic materials for a broad application. In contrast, application of chitosan, known as non-toxic biopolymer, can avoid these problems. As a renewable resource it has furthermore outstanding proper-ties such as biocompatibility176,177 as well as biodegradability178. Nevertheless it has to be considered that those properties only account for pure chitosan. It is well known that chitosan processed from chitin may contain heavy metals, protein residues as well as acid/alkali residues. Those impurities of the polymer, caused by the production process and the source of the preparation, can have an impact on man and the envi-ronment and have to be considered within the hazard assessment. In general chitosan preparations are obtained from natural chitin sources as for example (crab shells, squid pen, fungi) by an alkaline deacetylation which leads to varying chitosan preparations depending on process parameters. These preparations consist of a mixture of different chitosan entities with different molecular weight and different fraction of acetylation, which can limit some specific application. In contrast to this fact chitosan preparations
described in the literature or offered on the market are analytically highly undefined.
Mostly only few parameters of the set of possible analytical data characterizing a type of a chitosan preparation are given. For most of the commercial chitosan preparations parameters like weight-averaged molecular weight MW, polydispersity MW/MN, frac-tion of acetylafrac-tion FA, pattern of acetylation PA and impurity content (protein, heavy metal) are usually not known. To make chitosan preparations sustainable, especially for pharmaceutical applications, it becomes inevitably to obtain a reproducibility of product batches and this requires a reliable characterization of chitosan. Even if this analysis, facing a broad variety of characteristics, remains highly challenging it is in-dispensable for the above demanded quality standards as well as for a sound hazard assessment to analyze the risks for man and the environment.
Historically chitosan production is not accompanied by a detailed product analysis.
Therefore scientists and applicants barely find well defined products on the market.
Typically the obtained products are used as received. As a consequence scientific re-sults are difficult to compare and structure-activity or structure property-relationships can hardly be analyzed. To overcome this deficiency we propose the following procedure to improve the output of chitosan related scientific tests. First, a multi-dimensional experimental investigation is performed that is based on a theoretical analysis of chem-ical and physchem-ical structure variety of chitosan. The analysis allows to fully describe the sample mixture that depends strongly on origin of the raw precursor chitin and its preparation. Second, the given material is analyzed theoretically to allow for an identification of the variety of chitosan entities which can be all part of one single preparation. Third, drawn conclusions after combination of these steps are used to modify and improve existing production processes (if possible), interpret the effects on biological systems and yield in strategies to get more defined products for detailed scientific tests in the future.
Throughout this work we use the nomenclature proposed by the European Chitin Soci-ety (EUCHIS)179. Chitin and chitosan will be classified on the basis of their solubility and insolubility in 0.1 M acetic acid. Insoluble material is named chitin, soluble mate-rial is defined as chitosan. The different types of acetylation will be expressed as the mole fraction of acetylation FA. It is given in brackets after every distinct chitosan preparation e.g. a 10 % acetylated chitosan A will be written as Chi A [0.1].
Theoretical analysis: T-SAR based analysis of distinct chitosan entities A powerful tool for the understanding of properties and effects of chemicals is the ap-proach of SAR (thinking in terms of structure-activity-relationships). However, T-SAR usually deals with distinct chemical entities which can be described by one or more chemical structural formulas. In contrast, T-SAR used on polymeric chitosan has to deal always with mixtures of different chitosan entities. These entities can differ with respect to their chain-length, the degree of branching, fraction of acetylation (FA) and pattern of acetylation (PA). Fig. 7.1a-f shows different types of chemical entities which all can be part of a distinct chitosan preparation.
According to the systematic algorithm182 to be used in T-SAR to determine physical and chemical properties of a distinct chemical entity the different structural elements of chitosan entities will be theoretical analyzed now.
Completely acetylated chitin [1.0] consist of only one monomer (acetylglucosamine)
Primary structure Secondary structure Tertiary structure Quartary structure
aggregate AA
DD DA
D A
deacetylated unit acetylated unit
Extended 2/1 helix chitosan [0]
Relaxed 2/1-helix chitosan*HCl [0]
OH O R2
HO
R1 OH
O OH R2
HO
R1 OH
H H
d)
O R3
HO
NH2
OH
R2
e)
O R3
HO
NH3
OH
R2 Anion
O O
HO
NH2 OH
O OHO
NH
OH H3C
O
FA 1-FA
O O
HO
NH2 OH
O OHO
NH
OH H3C
O
n n
a) b)
c)
O O O
HO NH2 OH
O O
HO NH2
OH
O
OHO HO OH
O HO
OH OH
O
O
b-1,4 linked chitosan a-1,4 linked amylose
f)
g)
h)
glucosamin unit water molecule b®
a a
b glucosamin unit anion
®
b®
H-donor potential/H-bridge H-acceptor potential
Positive charge Negative charge
c
b®
O3
O5 O6
N2
a®
c O3 N2 O5
O6
i) j) k)
pka»6...6.5
O O
HO NH2
O OH
O O
HO NH
OH O O
HO NH2 OH
O HO
HO NH
OH
H3C O H3C
O
OH O O
HO NH
OH O O
HO NH2 OH
O HO
NH
OH
H3C O H3C
O
O O
HO NH
O OH
O O
HO NH
OH O O
HO NH OH
O HO
HO NH
OH
H3C O H3C
O
H3C
O H3C
O
OH O O
HO NH2
OH O O
HO NH2 OH
O HO
NH2
OH
FA= 0.57
FA= 0.57 PA= 2
PA= 0.34
Hairpin structure T 310K³
Extended structure T 310K£
a) b)
c)
d)
e)
500 nm
Figure 7.1: Structural diversity of chitosan- Chemical structure of polymers chitin [1.0](a) and chitosan [0.0](b). Both polymers are partially acetylated and appear as copolymers (c) characterized by an average fraction of acetylation FA. As sugar derivative chitin and chitosan possess a reducing end group function (aldehyde) (d). The amino group function of a deacetylated glucosamine unit shows a pH-dependant functionality (e). Chitin and chitosan can be distinguished by their pattern of acetylation PA even at identical FA (f). Chitin and chitosan are connected via 1→4 β-glycosidic linkages which leads to a stiffer chain in comparison to 1→4 α-linked sugars (g). As a macromolecule, chitosan has a
(Fig.7.1a). Its structure is comparable with murein and cellulose, which are main structural polymers creating the cell walls of bacteria (murein) and plants (cellulose).
Natural chitin chains consist of several thousands monomers associated to one another by very strong hydrogen-bonding between the amide nitrogen and the amide carbonyl groups of adjacent chains. For deacetylation of the amino group function (chitin) rela-tively strong reactants (concentrated alkali solutions) have to be applied to remove this less reactive group. If, for instance, all chitin monomers are deacetylated this will yield to ”ideal” chitosan [0.0], which consists of only glucosamine units (Fig.7.1b). Actually chitin and chitosan are both copolymers varying in chain length and FA (Fig.7.1c).
Every chitin/chitosan chain possesses a reducing end group (aldehyde function) which is a typical characteristic for sugars (Fig.7.1d). The ability of chitosan to dissolve in aqueous media is mainly derived from the protonation of the primary amino group function (Fig.7.1e). Occurring charges are one of the main factors for the dissolution process. However, this is constrained by the pKa of this group (pKa≈ 6 to 6.5). At alkaline conditions the charge inducing protonation disappears which leads to precipi-tation. Chitin, in contrast, has only a low percentage of free primary amino groups and is therefore not able to establish enough charges for a dissolution in aqueous media. In order to transform chitin into chitosan or vice versa an deacetylation or acetylation-process is necessary. Theoretically, this acetylation-process can lead to different patterns of the remaining acetyl groups at the polymeric chain (Fig.7.1f). For the same FA acetyl groups can possess a well ordered pattern (e.g. deacetylated (D) and acetylated (A) units alternate), a random pattern (e.g. D and A are randomly distributed along the chain) or a blockwise pattern (e.g. all A units can be found in one or more blocks on the chain) is possible. The monomers in chitin and chitosan are connected via (1→4) β-glycosidic linkages (Fig.7.1g). Thisβ-linkage restricts strongly the chain-mobility in solution. The polymer chain is constrained in its flexibility and therefore chitosan pos-sesses a relatively stiff chain resulting in huge intrinsic viscosity values in comparison toα-linked polysaccharides (e.g. amylose Fig.7.1g).
Being macromolecules the appearance of chitin and chitosan can be classified as pri-mary, secondary, tertiary and quartary structures. The primary structure of chitin and chitosan depends only on the two variable monomers: acetylglucosamine and glu-cosamine (Fig.7.1h). The sequence of these monomers will vary with respect to FA
and PA. Different secondary structures (Fig.7.1i) of solid chitosan crystallites were found by X-ray diffraction (XRD) and differing helical arrangement was revealed in the presence of different anions183. Chitosan shows an extended 2/1 helical structure, similar to chitin and cellulose. However, after crystallizing chitosan oligomer salts in the presence of different anions (e.g. nitrate, sulfate, chloride) the chain distorts and adopts a relaxed 2/1 helix (Fig.7.1i right). This structure is believed to be similar to the structure appearing after dissolution. An calculated tertiary structure was found by Sakajiri et al.181(Fig.7.1j). At higher temperatures (T≥ 310K) the extended 2/1 structure changes to a hairpin structure which is still stable even though the sample was cooled down again. The association to an ensemble of many polymers (quartary structure) is forced by intermolecular interactions (Fig.7.1k). Here a TEM photograph of a large chitosan aggregate is shown (Fig.7.1k).
Every chitosan preparation shows several of this fundamental properties. Some of them can influence the experimental analysis (solubility, aggregation, polymeric character) and are important to interpret the experimental data in the right way. Other param-eters (fraction of acetylation FA, pattern of acetylation PA, molecular weight MW)
may influence the effects on organisms more strongly and need to be analyzed before a detailed conclusion can be drawn. Within our approach we correlate physical and chemical properties of six selected chitosan preparations with its effects on two bio-logical test systems. This correlation represents a starting point for a more detailed investigation about the impact of chitosan entities on organisms. Hence, we want to focus on the necessity of a combined theoretical and experimental analysis representing the basis for an improved insight in structure-property and structure-activity relation-ships of chitosan.