Ph. D. Student

Atheer Jasim Mohammed/ MSc. Food EngineeringVisiting student – ITÜ

May, 2016

Antimicrobial polymers - Chitosan

Chitosan

Chitosan is a linear polysaccharide composed of randomly distributed β-(1-4)-linked D-glucosamine (de-acetylated unit) and N-acetyl-D-glucosamine (acetylated unit).

2Chemical structure of Chitosan. (C6H11O4N)n

Synthesis of Chitosan

De-acetylation of chitin includes the removal of CH3CO group

Chitosan is produced commercially by de-acetylation of chitin, which is the structural element in the exoskeletons of crustaceans.

Preparation of Chitosan

Properties of Chitosan

Biological properties of chitosan Physiochemical properties of chitosan Biocompatible Cationic polyamine

When protonated, adheres to negatively charged surfaces (bio/muco-adhesive) and forms gels with polyanions

Biodegradable Forms salts with organic and inorganic acids

Safe and non Toxic High molecular weight linear polyelectrolyte

Haemostatic, Bacteriostatic, and Fungi- static

Viscosity, high to low

Spermicidal Chelates certain transitional metals

Anticarcinogenic Amiable to chemical modification

Anticholesteremic Reactive amino/hydroxyl groups

High charge density at pH < 6.5

Applications of Chitosan

A. Daily Uses In Chemical Industries: It improves flocculation and is used in

filtration processes. It is also used as a subsidiary material of the color fixer,

patternizer, adhesive and stabilizer in plastic industry.

B. Agricultural Uses: Chitosan is used as seed coating and plant growth

enhancer.

C. Environmental Protection: Chitosan is used as active mud coagulant,

adhesive, adsorbent of the heavy metal ion and organic compound.

D. Biomedical Uses: Chitosan is used in making of bandages and hemostatic

agents due to its ability of clotting blood rapidly. Trimethyl chitosan is used to

transfect breast cancer cells and efficient in non viral gene delivery.

Aims of this study These chitosan thin films hold potential as low-cost, dissolvable bandages, or second skin, with antimicrobial properties that prohibit the most relevant intra hospital bacteria that infest burn injuries.

The mechanism of research

The mechanism of bacteria kill by chitosan films by the way like electrostatic interaction between NH3 groups and chitosan film and phosphoryl groups of phospholipid on cell membrane.

1. Film Preparation

A. Chitosan films were obtained by evaporation with organic acid solutions such as acetic and lactic acids, and also with different additives such as glycerol, fatty acids, and their mixtures.

B. The films were obtained from 25 mL of filtrated final mixture were placed in Petri dishes and placed in oven with air convection at 36 C for 12 h until complete film formation.

2. Film Characterization : Measured with an Electronic Digital Micrometer with a resolution of 1 mm. Thickness measurements and film morphology were also investigated using scanning electron microscopy (SEM).

3. Optical Spectroscopy of Chitosan Gel Films : The chitosan films were examined using Fourier transform infrared spectroscopy and spectroscopic ellipsometry at the wavelength range 400–900 nm.

MATERIALS AND METHODS

4. Nuclear Magnetic Resonance : with 3000 accumulations at a spinning frequency of 5.7 kHz.

5. Antimicrobial Assay : The antimicrobial activity of the films was measured by the diffusion disk method or Kirby–Bauer method to determine antimicrobial principle. Table 3 lists the 10 microorganisms investigated in the assays.

6. In Vitro Biodegradation Assay :

MATERIALS AND METHODS

The in vitro biodegradation of the films was measured under physiological conditions in a phosphate buffered solution.

ATCC: American Type Culture Collection

RESULTS AND DISCUSSION1. Figure1(a): Evaporation chitosan films have macro-scale

(1–100 mm) morphologies similar to cellophane and commercial polypropylenes.

2. Figure 1(c–e) show SEM examination of the surface and cross sections in the additives.

3. Figure 1(b) show morphology of gel films was smooth but contains pores that run through the thickness of the film from the top surface.

Results addition of glycerol and Tween 80 changes the characteristic.

The porosity in the chitosan composite mixtures during gelation and plasticizing allows oxygen permeation through a wound dressing, which maintains the healing rate and also the proliferation of anaerobic bacteria.

Atomic force microscopy measurements confirm that all additives, such as glycerol, give a more homogeneous, nonporous surface due to its plasticizing effect .

most uniform films, they are characterize and relate the differences in composition and structure to their antimicrobial and biodegradation properties.

Figure 2. FTIR spectrum of (a) chitosan powder and chitosan acetate and (b) chitosan composites which prepared according to experimental design..

Fourier Transform Infrared Spectroscopy FTIR1) FTIR spectra are shown in Figure 2(a,b), chitosan solution in acetic acid

without any additive, chitosan powder, and two representative chitosan films, are moved From 1400 to 1700 cm -1 in all samples, and find signals at 1656 cm -1 corresponding to the stretching of carbonyl group (C═O), and at 1598 cm -1 from the deformation mode of the free amino group NH2.

2) In the region 3000–3700 cm -1 [Figure 2(a,b)], all forms of the chitosan studied in this work display this band corresponding to OH stretching from hydration.

3) Separated signals of the OH groups at 3458 cm-1 and the NH band at ~3300 cm-1 confirm the reaction between the OH from chitosan and the acetic acid to form the ester group.

4) The bending mode from CH2 groups at 1432 cm-1, and CH3 groups at 1379 cm-1 in chitosan are blue-shifted to 1400 cm-1 in its polymeric derivative.

5) Figure 2(b) shows the FTIR spectra for A composites prepared, the band from stretching of the carbonyl group (C═O) is found at 1728 cm-1 and the stretching of the simple CO bond found between 1149 and 1018 cm-1

6) Bands observed at 1635 and 1603 cm-1 in pure chitosan are significantly changed in the spectra of their corresponding films. Bands attributed to C═O stretch (amide II) are shifted to lower frequency (1643 and 1557 cm-1) with respect to chitosan in those containing acetic acid, due to acetate presence.

7) gel films with lactic acid display a band from C═O group stretching at 1730 cm-1 in addition to C−C(C═O)−C mode stretches centered at 1250 cm-1 and O−C−C stretching at 1140 cm-1. These modes indicate the presence of chitosan lactate films, which is a critical step in the formation of antimicrobial films.

1) All spectra from the gels show signals from chitosan, in presence of carbonyl and methyl groups at 180 and 24 ppm.

2) Resonance at δ = 174.6 ppm related to N-acetyl glucosamine units is observed close to the resonance at 180 ppm which comes from carbonyl carbon from the carboxylate group.

3) NMR spectrum of gel obtained from chitosan solution in acetic acid with glycerol shows resonances of δ = 25 ppm in the spectrum, assigned to the CH3 carbon.

4) NMR signals from chitosan in the films are observed with resonance bands at δ = 104.7 (C4), δ = 85 (C3), δ = 74 (C5), δ = 60 (C6), δ = 56 (C2)

5) A very low intensity, wide peak from the film at ~120 ppm corresponds to remnant C═C bond presence. A significant presence of which is known to result in increased cytotoxicity.

6) At δ = 131.5 (C1) ppm, crosslinking with amino group during chitosan film formation is found.

Figure 3. NMR spectrum of acetate-Based gel film.

NMR

Antimicrobial and Biodegradation Properties of Chitosan Gel Films

Figure 4. (a) Diffusion disk results of chitosan composite films and (b) inhibition halo formation, against Klebsiella pneumonia (left) and Staphylococcus aureus (right).

1) Figure 4 shows the microorganisms are not observed to grow under the swollen film.

2) the contact area with the microorganism shows an inhibition of its growth in tandem with an increase is disk diameter.

3) The researchers are observed areas where there is negligible film growth even in the presence of microorganisms, which contradicts recent findings that report much higher (~98%) halo inhibition from films cast from solutions with a different pH.

4) The antimicrobial activity is limited or in some cases, not at all evident under certain preparatory conditions where the temperature exceeds 36C that result in dried thin films.

Antimicrobial and Biodegradation Properties of Chitosan Gel Films

almost all films reduce the number of colonies after 24 h of incubation by factors of ~105–107 CFU/mL, compared to controls.

Careful re-examination of ghost films shows that the antimicrobial efficacy is due to the chitosan and not from any residual acids or additives used in fabricating the films.

Despite the lack of FDA approval for chitosan, its potential for wound healing, controlled biodegradation, and drug delivery is promising.

Chitosan can be degraded in vitro by lysozyme, and its rate of degradation is inversely proportional to the degree of de-acetylation.

CONCLUSIONSA. chitosan gel films can be tuned with physical properties that have beneficial bactericidal and

antimicrobial effects in the control of the most relevant intra-hospital bacteria that infest burn injuries.

B. The correlation between preparation and resulting molecular structure was achieved through microscopy and spectroscopy investigations of the gel films .

C. The final biomaterial morphology and composition dependence of these films on the antimicrobial activity; films that undergo less biodegradation are acetates or lactates, smoothened by the choice of solvent and plasticizing additives to prevent cracking and porosity.

D. The corresponding antibacterial activity showed a relative dependence on the chitosan molecular weight, quantity, and solvent and typically allows a reduction in the number of colonies after 24 h of incubation by factors of ~105– 107 CFU/mL, compared with controls for some Gram-positive and Gram-negative bacteria.

E. As chitosan is a biomaterial already used in various medical devices and holds considerable potential in the fields of regenerative medicine and tissue engineering.

F. In tandem with its resistance to biodegradation gives a useful biopolymer while acknowledging the trade-off between resistance to enzymatic degradation and antimicrobial efficacy.

G. Thin gel films are capable of molding to bandages or second skin patches for burn-wound recovery.

for your attention …