**2. Modified chitosan membranes**

Modified chitosan was prepared through introducing extra amino groups to its structure. A new chemical route was used to graft selectively the extra amino groups on the hydroxyl groups of chitosan rather than its amine ones. To achieve this goal, chitin has been activated first with PBQ then amino groups were grafted using ethylene diamine as source of amino groups. The obtained aminated chitin was finally de-acetylated (21-22) to have modified chitosan with extra amino groups as shown in the following schema (figure 1). Different concentrations of PBQ were used to have modified chitosan with different amine content (table1). From the table it is clear that increasing PBQ concentration increased the nitrogen content in an obvious manner. This increment starts in linear way then turns to level off.

Fig. 1. Schematic diagram for synthesis of aminated chitosan.

ionized at pH below 6 and carry a positive charge. Unmodified chitosan is not antimicrobially active at pH 7, since it does not dissolve and also since it does not contain any positive charge on the amino groups (18, 19). The antimicrobial activity of chitosan also increases with increasing degree of deacetylation, due to the increasing number of ionisable amino groups (19). Several approaches were done to increase the antimicrobial activity of chitosan by introduce amino groups, on the primary amino groups of the back bone of chitosan polymer chains but it was failed (20). The obtained results were explained based on

In this work, we aim to increase both the solubility and antimicrobial activity of chitosan via increase the amino groups on the polymer back bone by attaching amino groups directly on

A new technique has been used to avoid the consumption of the original amino groups of the chitosan as sites of grafting, so chitin was first grafted with amino groups in separate step then it was de-acetylated to have the aminated chitosan. Aminated chitosan was tested as antimicrobial agent and aminated chitosan membranes were prepared, characterized and

Modified chitosan was prepared through introducing extra amino groups to its structure. A new chemical route was used to graft selectively the extra amino groups on the hydroxyl groups of chitosan rather than its amine ones. To achieve this goal, chitin has been activated first with PBQ then amino groups were grafted using ethylene diamine as source of amino groups. The obtained aminated chitin was finally de-acetylated (21-22) to have modified chitosan with extra amino groups as shown in the following schema (figure 1). Different concentrations of PBQ were used to have modified chitosan with different amine content (table1). From the table it is clear that increasing PBQ concentration increased the nitrogen content in an obvious manner. This increment starts

the remote position of the new introduced amino groups.

evaluated for wound dressing applications.

**2. Modified chitosan membranes** 

in linear way then turns to level off.

the hydroxyl groups of polysaccharide to wide its applications.

Fig. 1. Schematic diagram for synthesis of aminated chitosan.


Table 1. Effect of PBQ concentration on the nitrogen content of modified chitosan

#### **2.1 Characterization of modified chitosan membranes**

In the characterization of modified chitosan membranes, different characters were monitored to show the effect of modification process on their properties. The occurrence of amination process was verified through examination of the chemical structure changes using FT-IR, TGA analysis and solubility test.

The FTIR spectrum of the modified chitosan and intermediates to verify structure changes was obtained using FTIR-8400S SHIMDZU. Japan. As shown in figure 2, the major difference are the wide peaks at 3431 cm-1, (I) corresponding to the stretching vibration of – NH2 and OH groups became more sharp at modified chitosan as result of alternation of –OH groups with –NH2 groups. Absorption band intensity at 1560, 1649 cm-1 (II) corresponding to carbonyl bands have been increased in (AC ), curve (b), via introduce further carbonyl groups of PBQ as illustrated in schema1, then return to normal at aminated chitin, curve (c). Finally, peaks will reduced after deacetylation as a result of removal the acetyl groups in modified chitosan. This observation confirmed the occurrence of the modification process with different steps indicated in figure 1.

Fig. 2. FTIR spectra of chitin (a), activated chitin (AC) (b), aminated chitin (c) and modified chitosan (d).

Biopolymer Modifications for Biomedical Applications 359

Sample T50 Weight loss at

Aminated chitosan (0% glycerol) 325.89 19.638 % 42.47 % Aminated chitosan (2% glycerol) 333.06 16.523 % 43 % Aminated chitosan (4% glycerol) 346.72 14.415 % 42.68 % Aminated chitosan (6% glycerol) 335.86 17.741 % 42.57 %

Table 3. Thermo-gravimetric data of chitosan and aminated chitosan membranes with

role of the glycerol content between aminated chitosan chains.

increases by about 50% upon amination. This observation confirms the modification of membranes hydrophilicty as a result of the amination process. The second notice is the shift of T50, temperature needed to lose 50% of sample weight, to lower temperature which came as a result of chemical structure modification and as a result its hydophilicity improvement. The last notice is the insignificant changes of the characteristic thermo-gram of pristine chitosan in the temperature range 240 -380oC, which indicates the absence of crosslinking

The membrane was prepared as the following 1 gm of chitosan or aminated chitosan was dissolved in 50 ml 2% acetic acid. Glycerol was added to the solution as a plasticizer. The solution was strained through cheesecloth to remove any un-dissolved particulates. The solution was then casting in a clean Petri dish and left at room temperature for 48 hours to ensure complete solvent evaporation. The humidity of the room was not controlled. Once the membrane was dried and separated from the Petri dish it was rinsed with 500 ml of 1 M of NaOH. The rinsing of the membrane in a caustic solution gives the films water-resistance by neutralizing and removing any acetic acid anions present in the membrane then the membrane was washed with distilled water to remove the traces of alkali and neutralized it. Finally, the wet membranes were spread out and attached to the clean glass support with clamps and allowed to dray for 24 hours at room temperature. The resulting membranes

Physico-chemical properties like water uptake, tensile strength, elongation (23), surface roughness, SEM and finally water vapor permeability have been conducted for the casted membranes. The obtained results show the impact of modification process on the properties of chitosan membranes through evaluation of their bio-characters such as; antimicrobial

The introduction of biomaterials surface in blood creates a new interface between cellular and fluid components of blood and material. This results in a thermodynamic driving force that acts to reduce the solid-liquid interfacial free energy at this interface. Ignoring interactions with blood cellular components, the blood plasma-biomaterial interfacial free energy is a thermodynamic quantity that incorporates the surface free energy contributions of both solid and liquid phases and provides a measure of the driving force for the adsorption of blood components on solid surfaces. The configuration of the initially

activity (24-25), hemocompatibility (26), cytotoxicity (27), and biodegradability (28).

different percent of glycerol

were transparent and flexible.

**Water up-take** 

Chitosan 351.6 12.28 % 42.9 %

T (0-118) T (240-380)

Changes in the structure of modified chitosan via introducing extra hydrophilic amine groups were expected to influence its solubility. The solubility of chitosan and the modified chitosan in different pH was measured as in table 2. The improving of the solubility of modified chitosan over chitosan itself was attributed to grafted amino groups. The aminated chitosan solubility at pH range from 5.0 to 6.0 was almost double of its unmodified chitosan counter part. These results, along with the obtained data from FT-IR analysis, confirmed the occurrence of amination process.


Table 2. Solubility percent of chitosan and modified chitosan in different pH

Thermal graph metric analysis of chitosan and aminated chitosan membranes with different content of glycerol was done; figures 3. The data of the TGA was summarized in table (3). From the table a number of informations were abstracted. First notice is the increase of weight loss in the temperature range 0 – 118oC which refers to loss of water. The water loss

Fig. 3. Thermal graph metric diagrams of chitosan membrane (a), chitosan modified membrane (b), chitosan modified membrane (glycerol 2%)(c), chitosan modified membrane (glycerol 4%)(d), and chitosan modified membrane (glycerol 6%)(e).

Changes in the structure of modified chitosan via introducing extra hydrophilic amine groups were expected to influence its solubility. The solubility of chitosan and the modified chitosan in different pH was measured as in table 2. The improving of the solubility of modified chitosan over chitosan itself was attributed to grafted amino groups. The aminated chitosan solubility at pH range from 5.0 to 6.0 was almost double of its unmodified chitosan counter part. These results, along with the obtained data from FT-IR analysis, confirmed the

pH Chitosan solubility (%) Modified chitosan solubility (%)

Thermal graph metric analysis of chitosan and aminated chitosan membranes with different content of glycerol was done; figures 3. The data of the TGA was summarized in table (3). From the table a number of informations were abstracted. First notice is the increase of weight loss in the temperature range 0 – 118oC which refers to loss of water. The water loss

Fig. 3. Thermal graph metric diagrams of chitosan membrane (a), chitosan modified

(glycerol 4%)(d), and chitosan modified membrane (glycerol 6%)(e).

membrane (b), chitosan modified membrane (glycerol 2%)(c), chitosan modified membrane

4 97.2 99 5 41.7 81.1 6 17.9 29.6 7 0 3.9 8 0 0 Table 2. Solubility percent of chitosan and modified chitosan in different pH

occurrence of amination process.


Table 3. Thermo-gravimetric data of chitosan and aminated chitosan membranes with different percent of glycerol

increases by about 50% upon amination. This observation confirms the modification of membranes hydrophilicty as a result of the amination process. The second notice is the shift of T50, temperature needed to lose 50% of sample weight, to lower temperature which came as a result of chemical structure modification and as a result its hydophilicity improvement. The last notice is the insignificant changes of the characteristic thermo-gram of pristine chitosan in the temperature range 240 -380oC, which indicates the absence of crosslinking role of the glycerol content between aminated chitosan chains.

The membrane was prepared as the following 1 gm of chitosan or aminated chitosan was dissolved in 50 ml 2% acetic acid. Glycerol was added to the solution as a plasticizer. The solution was strained through cheesecloth to remove any un-dissolved particulates. The solution was then casting in a clean Petri dish and left at room temperature for 48 hours to ensure complete solvent evaporation. The humidity of the room was not controlled. Once the membrane was dried and separated from the Petri dish it was rinsed with 500 ml of 1 M of NaOH. The rinsing of the membrane in a caustic solution gives the films water-resistance by neutralizing and removing any acetic acid anions present in the membrane then the membrane was washed with distilled water to remove the traces of alkali and neutralized it.

Finally, the wet membranes were spread out and attached to the clean glass support with clamps and allowed to dray for 24 hours at room temperature. The resulting membranes were transparent and flexible.

Physico-chemical properties like water uptake, tensile strength, elongation (23), surface roughness, SEM and finally water vapor permeability have been conducted for the casted membranes. The obtained results show the impact of modification process on the properties of chitosan membranes through evaluation of their bio-characters such as; antimicrobial activity (24-25), hemocompatibility (26), cytotoxicity (27), and biodegradability (28).

#### **Water up-take**

The introduction of biomaterials surface in blood creates a new interface between cellular and fluid components of blood and material. This results in a thermodynamic driving force that acts to reduce the solid-liquid interfacial free energy at this interface. Ignoring interactions with blood cellular components, the blood plasma-biomaterial interfacial free energy is a thermodynamic quantity that incorporates the surface free energy contributions of both solid and liquid phases and provides a measure of the driving force for the adsorption of blood components on solid surfaces. The configuration of the initially

Biopolymer Modifications for Biomedical Applications 361

Aminated chitosan (0% glycerol) 20.60 N 31.85 % 43.32 Aminated chitosan (2% glycerol) 26.88 N 28.60 % 46.66 Aminated chitosan (4% glycerol) 25.78 N 29.30 % 36.83 Aminated chitosan (6% glycerol) 28.75 N 34.33 % 42.91 Table 5. The maximum stress and strain of chitosan and aminated chitosan membranes with

rigidity of native chitosan. Other authors used poly propylene glycol as a plasticizer and obtained similar results. They claimed the obtained results to the fact that propylene glycol is containing several OH groups, which can form intermolecular bonds with the excess of

It is known that biomaterials have to fulfill many conditions. However, the surface quality is one of the most important properties of biomaterial, which limits its applications. It is so important because most of biological reactions occur on the surface and at interface. Among other things the hydrophilicty, chemical structure, topography and roughness of surface create the response of the host tissue to presence of implant. The adhesion and proliferation of cells is also determined by the surface properties. They play a significant role in

The surface roughness of chitosan and aminated chitosan membranes was evaluated (table 6). It was found that the roughness of aminated chitosan membranes is less than chitosan. Also the table shows a decrease in the film roughness with increase of glycerol concentration. This behavior could be referred to the changes in chemical structure, hydrophilicty and as well as the surface energy which separately or in combination affect the arrangement of the macromolecules and hence its surface roughness. It can be seen the role of glycerol in improving the surface smoothness. In natural skin, the dense skin layer has a surface roughness of 20 to 165 nm. This means that increase of the glycerol

Sample Surface roughness µm

Aminated chitosan (0% glycerol) 2.34 2.28 2.31 Aminated chitosan (2% glycerol) 1.92 1.3 1.611 Aminated chitosan (4% glycerol) 1.73 1.25 1.49 Aminated chitosan (6% glycerol) 1.4 1.28 1.35 Table 6. Surface roughness of chitosan and aminated chitosan membranes with different

Chitosan 3.853 3.57 3.7133

Front face Behind face average

N

Chitosan 19.80 N 24.36 37.72

Max strain λmax %

Max stress σmax N/mm2

Sample Max force

different percent of glycerol

**Surface roughness** 

percent of glycerol

NH3+ groups in the chitosan polymer (32).

biocompatibility as well as for tissue engineering.

adsorbed proteins on the solid surface may be determined by the magnitude of the blood plasma-biomaterial interfacial free energy (29). Andrade proposed the minimum interfacial free energy hypothesis of biocompatibility (30). Water plays an important role in determining the biocompatibility of synthetic materials. Ratner et al. (31) have recognized that high water levels within the surface of materials will help provide a low interfacial free energy with blood and will reduce both protein adsorption and cell adhesion on the polymeric surface. Therefore, a surface with a hydrated polymer (hydrogel) coating would be expected to be more compatible with body fluids than a non-polar or less hydrated type of surface. The water up take of chitosan and aminated chitosan membranes with 0, 2, 4 and 6 % glycerol content was recorded in table (4).


Table 4. Water uptake of chitosan and aminated chitosan membranes with different percent of glycerol

The water sorption of the chitosan was attributed to the hydrophilic groups of the polysaccharide chains; hydroxyl and amino groups. From the table, it is clear that increase of the water sorption of aminated chitosan over the chitosan it self. This was explained by increase the hydrophilic groups on the chitosan via grafted with amine groups. In the other hand, the increase of the water sorption of the aminated chitosan as increase the plasticizer percent was attributed to the hydrophilic power of the glycerol as polyols and also results from the effect of the plasticizer on the limitation of crystallinty of the membrane. The obtained results are in accordance with published results by other authors in which they added poly propylene glycol as a plasticizer to chitosan (32).

#### **Mechanical properties**

The tensile properties of chitosan and aminated chitosan membranes with different percent of plasticizer were measured and recorded in table (5). It was determined from the critical breaking point of the stretching test pieces. The maximum stress σmax (Nm−2) was evaluated as the ratio of the stretching force divided by the cross-sectional area of broken membrane piece. The maximum strain λmax was measured as the elongation ratio of the initial length of the test piece.

The effect of the stress on the elongation of the membranes was found more clear for aminated chitosan higher than chitosan. The change of elastic intensity suggested that the number of the functional groups significantly affected the network elasticity of chitosan. This was reflected on increase the maximum stress and decreases the maximum strain.

By added the glycerol, the elongation of the aminated chitosan was increased with increase the percent of glycerol in polymer. Glycerol is miscible easily with chitosan, so the introduction of glycerol moisture resulting in drastic chain flexibility which reduced the


Table 5. The maximum stress and strain of chitosan and aminated chitosan membranes with different percent of glycerol

rigidity of native chitosan. Other authors used poly propylene glycol as a plasticizer and obtained similar results. They claimed the obtained results to the fact that propylene glycol is containing several OH groups, which can form intermolecular bonds with the excess of NH3+ groups in the chitosan polymer (32).

#### **Surface roughness**

360 Infrared Spectroscopy – Life and Biomedical Sciences

adsorbed proteins on the solid surface may be determined by the magnitude of the blood plasma-biomaterial interfacial free energy (29). Andrade proposed the minimum interfacial free energy hypothesis of biocompatibility (30). Water plays an important role in determining the biocompatibility of synthetic materials. Ratner et al. (31) have recognized that high water levels within the surface of materials will help provide a low interfacial free energy with blood and will reduce both protein adsorption and cell adhesion on the polymeric surface. Therefore, a surface with a hydrated polymer (hydrogel) coating would be expected to be more compatible with body fluids than a non-polar or less hydrated type of surface. The water up take of chitosan and aminated chitosan membranes with 0, 2, 4 and

Aminated chitosan (glycerol 0%) 197.83 Aminated chitosan (glycerol 2%) 208.94 Aminated chitosan (glycerol 4%) 220.62 Aminated chitosan (glycerol 6%) 255.59 Table 4. Water uptake of chitosan and aminated chitosan membranes with different percent

The water sorption of the chitosan was attributed to the hydrophilic groups of the polysaccharide chains; hydroxyl and amino groups. From the table, it is clear that increase of the water sorption of aminated chitosan over the chitosan it self. This was explained by increase the hydrophilic groups on the chitosan via grafted with amine groups. In the other hand, the increase of the water sorption of the aminated chitosan as increase the plasticizer percent was attributed to the hydrophilic power of the glycerol as polyols and also results from the effect of the plasticizer on the limitation of crystallinty of the membrane. The obtained results are in accordance with published results by other authors in which they

The tensile properties of chitosan and aminated chitosan membranes with different percent of plasticizer were measured and recorded in table (5). It was determined from the critical breaking point of the stretching test pieces. The maximum stress σmax (Nm−2) was evaluated as the ratio of the stretching force divided by the cross-sectional area of broken membrane piece. The maximum strain λmax was measured as the elongation ratio of the initial length of

The effect of the stress on the elongation of the membranes was found more clear for aminated chitosan higher than chitosan. The change of elastic intensity suggested that the number of the functional groups significantly affected the network elasticity of chitosan. This was reflected on increase the maximum stress and decreases the maximum strain.

By added the glycerol, the elongation of the aminated chitosan was increased with increase the percent of glycerol in polymer. Glycerol is miscible easily with chitosan, so the introduction of glycerol moisture resulting in drastic chain flexibility which reduced the

Sample Water uptake % Chitosan 183.14

6 % glycerol content was recorded in table (4).

added poly propylene glycol as a plasticizer to chitosan (32).

of glycerol

**Mechanical properties** 

the test piece.

It is known that biomaterials have to fulfill many conditions. However, the surface quality is one of the most important properties of biomaterial, which limits its applications. It is so important because most of biological reactions occur on the surface and at interface. Among other things the hydrophilicty, chemical structure, topography and roughness of surface create the response of the host tissue to presence of implant. The adhesion and proliferation of cells is also determined by the surface properties. They play a significant role in biocompatibility as well as for tissue engineering.

The surface roughness of chitosan and aminated chitosan membranes was evaluated (table 6). It was found that the roughness of aminated chitosan membranes is less than chitosan. Also the table shows a decrease in the film roughness with increase of glycerol concentration. This behavior could be referred to the changes in chemical structure, hydrophilicty and as well as the surface energy which separately or in combination affect the arrangement of the macromolecules and hence its surface roughness. It can be seen the role of glycerol in improving the surface smoothness. In natural skin, the dense skin layer has a surface roughness of 20 to 165 nm. This means that increase of the glycerol


Table 6. Surface roughness of chitosan and aminated chitosan membranes with different percent of glycerol

Biopolymer Modifications for Biomedical Applications 363

The decrease of the membrane permeability as exposed to water vapor was attributed to two factors. First, the swelling of polymer chains which decreases the pores diameter of the membrane and second is the reduction of the pore volume due to the presence of bulk PBQ benzene ring in the structure. The decrease of the aminated chitosan membranes permeability proved the changes in the chemical structure which in consequence affects the hydrophilicity of the membranes. In the same way, the addition of glycerol decreases the permeability of membranes gradually with its content. These results are in accordance with the water uptake percentage measurements. This future enables us to control the water

For application as wound dressing, modified chitosan membranes have been evaluated from Bio-point of view. Properties such as cytotoxicity, Hemocompatability and

Chitosan derivatives present interesting properties for biomedical applications, because such materials can exhibit enhanced bacteriostatic activity with respect to pure chitosan. Ethylene diamine tetraacetic acid (EDTA) grafted onto chitosan increases the antibacterial activity of chitosan by complexing magnesium that under normal circumstances stabilizes the outer membrane of gram-negative bacteria (33). The increase in chitosan antimicrobial activity is also observed with carboxymethyl chitosan, which makes essential transition metal ions unavailable for bacteria (34) or binds to the negatively charged bacterial surface to disturb the cell membrane (35). Therefore, these materials are used in wound healing systems, such as carboxymethyl chitosan for the reduction of periodontal pockets in dentistry (34) and chitosan-grafted with EDTA as a constituent of hydro-alcoholic gels for topical use (33). Chitosan and chitooligosaccharide-grafted membranes showed antibacterial activity against *Escherichia coli*, *Pseudomonas aeruginosa*, methicilin-resistant *Staphylococcus aureus* (MRSA), and *S. aureus* (36). Also, it was observed that the antimicrobial activity of chitosan and graft copolymers against *Candida albicans*, *Trichophyton rubrum*, and *Trichophyton violaceum* depends largely on the amount and type of grafted chains, as well as

Table 8 shows the effect of chitosan amination degree variation on its antimicrobial activity. It was found that antimicrobial activity of the aminated chitosan increased as a result of increase grafted amine groups due to selective grafting of this external amine groups on the hydroxyl groups of chitosan. This leads consequently to increase the positive charges on the polymer backbone. The antibacterial activity was improved against two gram negative bacteria, *Escherichia coli* and *pseudomonas aeruginosa*, by 50% and 40%, respectively. At the same time, it was improved against two gram positive bacteria, *Bacillus cereus* and *Staphylococcus aureus*, by 114% and 45%, respectively. In microbial profile, the powerful effect of modified chitosan on the gram negative rather than the gram positive bacterium was explained by the difference on the pathological composition of the cell wall. Gram negative bacteria have thick layer of phospholipids rather than the peptidoglycan comparing to the gram positive which has thick layer of peptidoglycan. The negative charges of the phospholipids enhance the adhesion power of poly cationic polymer such as

vapor permeability.

**Antimicrobial activity** 

on the changes of pH (37).

**2.2 Bio-evaluation of modified chitosan** 

Biodegradability have been evaluated.

concentration in the membrane caste solution, brings the membrane roughness close to that of skin which increases the contact area and hence preventing frictions and simultaneously reduces the healing time.

#### **Scanning electron microscope**

The surface of chitosan membrane and aminated chitosan membranes with different percent of plasticizer was scanned using scan electron microscope (SEM) (figure 4). The surface became smoother with amination and furthermore with glycerol addition. This observation is in agreement with roughness test obtained data in table 6.

Fig. 4. Surface scan electron microscope photo of chitosan membrane (a), aminated chitosan membrane (b), aminated chitosan membrane with 2% glycerol (c), aaminated chitosan membrane 12 with 4% glycerol (d), and aminated chitosan membrane with 6% glycerol (e).

#### **Water vapour permeability**

One of the main functions of wound dressing membrane is maintains a moist environment at the wound/ dressing interface. This characteristic was measured for chitosan and modified chitosan membranes, with different glycerol content, through measuring of its water vapor permeability (table 7).


Table 7. Water permeability of chitosan and aminated chitosan membranes with different percent of glycerol

The decrease of the membrane permeability as exposed to water vapor was attributed to two factors. First, the swelling of polymer chains which decreases the pores diameter of the membrane and second is the reduction of the pore volume due to the presence of bulk PBQ benzene ring in the structure. The decrease of the aminated chitosan membranes permeability proved the changes in the chemical structure which in consequence affects the hydrophilicity of the membranes. In the same way, the addition of glycerol decreases the permeability of membranes gradually with its content. These results are in accordance with the water uptake percentage measurements. This future enables us to control the water vapor permeability.

#### **2.2 Bio-evaluation of modified chitosan**

For application as wound dressing, modified chitosan membranes have been evaluated from Bio-point of view. Properties such as cytotoxicity, Hemocompatability and Biodegradability have been evaluated.

#### **Antimicrobial activity**

362 Infrared Spectroscopy – Life and Biomedical Sciences

concentration in the membrane caste solution, brings the membrane roughness close to that of skin which increases the contact area and hence preventing frictions and simultaneously

The surface of chitosan membrane and aminated chitosan membranes with different percent of plasticizer was scanned using scan electron microscope (SEM) (figure 4). The surface became smoother with amination and furthermore with glycerol addition. This observation

Fig. 4. Surface scan electron microscope photo of chitosan membrane (a), aminated chitosan membrane (b), aminated chitosan membrane with 2% glycerol (c), aaminated chitosan membrane 12 with 4% glycerol (d), and aminated chitosan membrane with 6% glycerol (e).

One of the main functions of wound dressing membrane is maintains a moist environment at the wound/ dressing interface. This characteristic was measured for chitosan and modified chitosan membranes, with different glycerol content, through measuring of its

Sample Permeability

Chitosan 3.843057

Aminated chitosan (glycerol 0%) 3.372898 Aminated chitosan (glycerol 2%) 2.476318 Aminated chitosan (glycerol 4%) 1.178036 Aminated chitosan (glycerol 6%) 0.586429

Table 7. Water permeability of chitosan and aminated chitosan membranes with different

gm – mil / m2-day

reduces the healing time.

**Scanning electron microscope** 

**Water vapour permeability** 

percent of glycerol

water vapor permeability (table 7).

is in agreement with roughness test obtained data in table 6.

Chitosan derivatives present interesting properties for biomedical applications, because such materials can exhibit enhanced bacteriostatic activity with respect to pure chitosan. Ethylene diamine tetraacetic acid (EDTA) grafted onto chitosan increases the antibacterial activity of chitosan by complexing magnesium that under normal circumstances stabilizes the outer membrane of gram-negative bacteria (33). The increase in chitosan antimicrobial activity is also observed with carboxymethyl chitosan, which makes essential transition metal ions unavailable for bacteria (34) or binds to the negatively charged bacterial surface to disturb the cell membrane (35). Therefore, these materials are used in wound healing systems, such as carboxymethyl chitosan for the reduction of periodontal pockets in dentistry (34) and chitosan-grafted with EDTA as a constituent of hydro-alcoholic gels for topical use (33). Chitosan and chitooligosaccharide-grafted membranes showed antibacterial activity against *Escherichia coli*, *Pseudomonas aeruginosa*, methicilin-resistant *Staphylococcus aureus* (MRSA), and *S. aureus* (36). Also, it was observed that the antimicrobial activity of chitosan and graft copolymers against *Candida albicans*, *Trichophyton rubrum*, and *Trichophyton violaceum* depends largely on the amount and type of grafted chains, as well as on the changes of pH (37).

Table 8 shows the effect of chitosan amination degree variation on its antimicrobial activity. It was found that antimicrobial activity of the aminated chitosan increased as a result of increase grafted amine groups due to selective grafting of this external amine groups on the hydroxyl groups of chitosan. This leads consequently to increase the positive charges on the polymer backbone. The antibacterial activity was improved against two gram negative bacteria, *Escherichia coli* and *pseudomonas aeruginosa*, by 50% and 40%, respectively. At the same time, it was improved against two gram positive bacteria, *Bacillus cereus* and *Staphylococcus aureus*, by 114% and 45%, respectively. In microbial profile, the powerful effect of modified chitosan on the gram negative rather than the gram positive bacterium was explained by the difference on the pathological composition of the cell wall. Gram negative bacteria have thick layer of phospholipids rather than the peptidoglycan comparing to the gram positive which has thick layer of peptidoglycan. The negative charges of the phospholipids enhance the adhesion power of poly cationic polymer such as

Biopolymer Modifications for Biomedical Applications 365

Aminated chitosan (glycerol 0%) 2.109617 Aminated chitosan (glycerol 2%) 1.447777 Aminated chitosan (glycerol 4%) 1.168563 Aminated chitosan (glycerol 6%) 1.013444 Table 9. Hemolytic percent of chitosan and aminated chitosan membranes with different

heamolysis to less than 2%, so the presence of glycerol is beneficial from this point of view. Best results obtained with 6% sample; 1.01 hemolytic percent. The effect of glycerol addition may be explained according to reduce the surface roughness and accordingly the contact area between the blood vessels and the membranes surface. It is worthy to mention here that the reduction of hemolytic effect is directly proportional with surface roughness mentioned in table (6). Reduction of surface roughness by 50% relative to chitosan has been obtained with aminated chitosan with 2% glycerol. This reduction was aligned with reduction in hemolytic percentage by about 25%. Critical value of surface roughness obtained with aminated chitosane with 2% glycerol, 1.61, which beyond the hemolytic percent reduced linearly and with higher rate. Addition of glycerol as plastizer may also contribute in

Several searches were tested and measured the cytotoxicity of Chitosan (39, 42). Cytotoxicity of Chitosan and aminated Chitosan was measured using mouse fibroblasts with the direct connect method and the data are shown in table 10. According to the tabulated results, the

> Dead cell X 105

Control 6.75 1.2 7.95 89.9 Chitosan 5.35 0.8 6.15 86.9 aminated Chitosan 5 1.5 6.5 76.9 4.3 1.6 5.9 72.9 aminated Chitosan with

Table 10. Cytotoxicity of chitosan and modified chitosan (0.6 x 105 cells incubated for five days)

Several authors have discussed the role of amine substitute on polymers in influencing toxicity. Dekie et al. (43) noted that the presence of primary amines in poly(L-glutamic acid) (PGA) derivatives has a significant toxic effect on red blood cells. Based on studies with modified poly(L-lysin) (PLL), Ferruti et al, (44) conclude that polymers with tertiary amine groups exhibit a lower toxicity than those with primary and secondary residues. They have also synthesized tertiary amine group containing poly (amidoamine)s (PAAS); these substitutes polymers have good biocompatibility and can form complexes with heparin

viability %

Total cells X 105

viability of the live cell decreased by increase the amine group substitution.

Live cells

X 105 sample

percent of glycerol

**Cytotoxicity** 

increasing of the porous structure.

plasticizer glycerol (4% )

Sample Hemolytic percent Chitosan 1.933816


Table 8. Effect of the degree of amination on the antimicrobial activity of modified chitosan against different microorganisms

chitosan on the cell wall. Increasing the cell inhibition capability of modified aminated chitosan affects directly on the time needed for wound healing and almost eliminates the possibility of bacterial infections. This idea was the base for preparation and synthesis of different quaternary chitosan derivatives (38). Improvement of the antibacterial activity has been achieved by other authors by adding silver to chitosane polyphosphate membranes (39). However, the cytotoxicity of this derivative was found very high.

The effect of adding glycerol as plasticizer to aminated chitosan on the antimicrobial activity was studied. Four samples with different percent of glycerol 0, 2, 4 and 6 % were tested. It was found that the antibacterial activity is nearly constant and does not affect with increase of the glycerol content in the membranes.

#### **Hemocompatability**

Hemolysis is regarded as an especially significant screening test, once it provides quantification of small levels of plasma hemoglobin, which may not be measurable under in vivo conditions. As reported in literature (ISO 10993–4 (1999)), it is not possible to define a universal level of acceptable or unacceptable amounts of hemolysis. Although by definition a blood-compatible material should be non-hemolytic, in practice several medical devices cause hemolysis. This means that when such hemolytic effect takes place, it is important to make sure that clinical benefits overcome the risks and that the values of hemolysis are within acceptable limits. It has been shown that chitosan derivatives have great potential to be used in other biomedical applications. As a result of the biocompatible properties such as good blood compatibility and cell growth efficiency, grafted chitosan materials have potential to be used in cardio-vascular applications (40-41).

According to ASTM F 756-00 (2000) materials can be classified in three different categories according to their hemolytic index (hemolysis %). Materials with percentages of hemolysis over 5% are considered hemolytic; while the ones with hemolytic index between 5% and 2% are classified as slightly hemolytic. Finally, when the material presents a hemolysis percentage below 2% it is considered as a non-hemolytic material. Table 9 represents the values of hemolysis obtained for all membranes. The obtained results indicate that the chitosan and chitosan derivatives are non hemolytic compounds and compatible with human body. Addition of glycerol with any percent of studied range reduced the


Table 9. Hemolytic percent of chitosan and aminated chitosan membranes with different percent of glycerol

heamolysis to less than 2%, so the presence of glycerol is beneficial from this point of view. Best results obtained with 6% sample; 1.01 hemolytic percent. The effect of glycerol addition may be explained according to reduce the surface roughness and accordingly the contact area between the blood vessels and the membranes surface. It is worthy to mention here that the reduction of hemolytic effect is directly proportional with surface roughness mentioned in table (6). Reduction of surface roughness by 50% relative to chitosan has been obtained with aminated chitosan with 2% glycerol. This reduction was aligned with reduction in hemolytic percentage by about 25%. Critical value of surface roughness obtained with aminated chitosane with 2% glycerol, 1.61, which beyond the hemolytic percent reduced linearly and with higher rate. Addition of glycerol as plastizer may also contribute in increasing of the porous structure.

#### **Cytotoxicity**

364 Infrared Spectroscopy – Life and Biomedical Sciences

Chitosan control 100 62.6298 35.689 68.73178 67.047076 0.0935 101 77.4394 46.2014 80.53936 69.258203 0.187 102 79.8616 57.3322 86.22449 73.18117 0.374 117.7 83.1834 58.2155 87.17201 85.805991 0.545 129.3 88.7889 62.3675 92.34694 94.293866 0.747 129.6 90.7958 76.7668 96.13703 97.360913

Table 8. Effect of the degree of amination on the antimicrobial activity of modified chitosan

chitosan on the cell wall. Increasing the cell inhibition capability of modified aminated chitosan affects directly on the time needed for wound healing and almost eliminates the possibility of bacterial infections. This idea was the base for preparation and synthesis of different quaternary chitosan derivatives (38). Improvement of the antibacterial activity has been achieved by other authors by adding silver to chitosane polyphosphate membranes

The effect of adding glycerol as plasticizer to aminated chitosan on the antimicrobial activity was studied. Four samples with different percent of glycerol 0, 2, 4 and 6 % were tested. It was found that the antibacterial activity is nearly constant and does not affect with increase

Hemolysis is regarded as an especially significant screening test, once it provides quantification of small levels of plasma hemoglobin, which may not be measurable under in vivo conditions. As reported in literature (ISO 10993–4 (1999)), it is not possible to define a universal level of acceptable or unacceptable amounts of hemolysis. Although by definition a blood-compatible material should be non-hemolytic, in practice several medical devices cause hemolysis. This means that when such hemolytic effect takes place, it is important to make sure that clinical benefits overcome the risks and that the values of hemolysis are within acceptable limits. It has been shown that chitosan derivatives have great potential to be used in other biomedical applications. As a result of the biocompatible properties such as good blood compatibility and cell growth efficiency, grafted chitosan materials have

According to ASTM F 756-00 (2000) materials can be classified in three different categories according to their hemolytic index (hemolysis %). Materials with percentages of hemolysis over 5% are considered hemolytic; while the ones with hemolytic index between 5% and 2% are classified as slightly hemolytic. Finally, when the material presents a hemolysis percentage below 2% it is considered as a non-hemolytic material. Table 9 represents the values of hemolysis obtained for all membranes. The obtained results indicate that the chitosan and chitosan derivatives are non hemolytic compounds and compatible with human body. Addition of glycerol with any percent of studied range reduced the

(39). However, the cytotoxicity of this derivative was found very high.

potential to be used in cardio-vascular applications (40-41).

Maximum inhibition (%)

*E.coli Bacillus Pseudomonas Stapylococcus*

PBQ :chitin ratio

against different microorganisms

of the glycerol content in the membranes.

**Hemocompatability** 

N content (relative percentage)

> Several searches were tested and measured the cytotoxicity of Chitosan (39, 42). Cytotoxicity of Chitosan and aminated Chitosan was measured using mouse fibroblasts with the direct connect method and the data are shown in table 10. According to the tabulated results, the viability of the live cell decreased by increase the amine group substitution.



Several authors have discussed the role of amine substitute on polymers in influencing toxicity. Dekie et al. (43) noted that the presence of primary amines in poly(L-glutamic acid) (PGA) derivatives has a significant toxic effect on red blood cells. Based on studies with modified poly(L-lysin) (PLL), Ferruti et al, (44) conclude that polymers with tertiary amine groups exhibit a lower toxicity than those with primary and secondary residues. They have also synthesized tertiary amine group containing poly (amidoamine)s (PAAS); these substitutes polymers have good biocompatibility and can form complexes with heparin

Biopolymer Modifications for Biomedical Applications 367

In conclusion the new route of modification was found successful and is recommended for

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(45-47). Fischer et al (48) confirmed these observations for PLL and PEI, but argued that cationized human serum albumin and starburst dendimer, which also contain primary amino groups, showed only moderate cytotoxic effects. They conclude that not only the type of amino function but also the charge density and arrangement is an important factor for determining cytotoxicity and hence biocompatibility (48). This may be explains the moderate increment of cytotoxicity of modified chitosan, table 10, in which the cell viability % decreases by about 10%, although the nitrogen content increases by about 30%. Only part of this increment is referred to primary amino groups and the other part is referred to secondary amino groups (figure 1), which is less toxic.

#### **Biodegradability**

The biodegradability of chitosan and aminated chitosan was measured as produced reduced sugar results from the cellulase enzyme action on the chitosan. Table 11 represents the OD of the total reduced sugar produced from chitosan and aminated chitosan hydrolysis with cellulase enzyme for 30 minutes at 50oC. It was observed that the degradation in the case of chitosan is higher than the aminated chitosan. This may be attributed to the steric effect result from increase the amine content and presence of benzene ring of PBQ on the polymer chains which consequently reduced the accessibility of the aminated chitosan to enzyme hydrolytic activity.


Table 11. Biodegradability effect of cellulase enzyme on the chitosan and aminated chitosan (30 minute, at 50oC and pH 7)
