β-Chitin/Chitosan Obtained from Loligo and Humboldt Squids Effectiveness as Wound Dressing Material

JINDAN Rahim ，ZALAL Rahim，HUDSON Samuel，KING W Martin，2

1 College of Textiles，North Carolina State University，Raleigh NC 27695，USA

2 College of Textiles，Donghua University，Shanghai 201620，China

Introduction

Metallic ions utilized in wound dressings and wound care scaffolds provide antibacterial activity but metallic particles in ionic form such as silver and zinc have been reported to cause toxic effects if released into the body over extended periods of time［1］and in some instances have led to other chronic diseases.Natural materials，such as chitin—a polysaccharide found in nature，are utilized for a number of biomedical applications.Chitin can be found in three different structural forms α，β，and γ.Previous studies on these structures of chitin and chitosan have shown that β-chitin can be dissolved in mild aqueous solvents without the use of harsh organic chemical compounds as solvents［2］.This approach is suitable for the study of biomaterials as the solvents used can be subsequently completely removed，leaving the biomaterial safe to use.β-chitin has also shown to have better bioresorption compared with the other forms of chitin/chitosan.β-chitin/chitosan is found in squids and tube worms which grow around hydraulic vents.The chemical structure for β-chitin is examined in terms of the intrinsic viscosity，pH，and infrared spectroscopy for pens of Humboldt and Loligo squids.The intrinsic viscosity and pH measurements are important for materials derived from natural resources.It helps determine difference between different natural resources.Intrinsic viscosity measurements are also useful to determine average molecular weight of the dilute polymer solutions using Mark-Houwink-Sakuada relationship［3］.While the Fourier transformed infra-red spectroscopy (FTIR)is a qualitative method for qualitative chemical analysis used to confirm the presence of various chemical groups within and near the surface of the materials.

1 Materials and Methods

The Humboldt squid pen samples were provided by Hopkins Marine Station of Stanford University obtained from the Bay of California，while the Loligo squid pens were obtained from the Department of Molecular and Cellular Biology at Harvard University which had harvested them from the Gulf of Thailand.Calcium chloride dihydrate，methanol，and acetic acid were purchased from Sigma Aldrich.Specta/Por 1 ®dialysis membrane tubing with molecular weight cut off(MWCO of 6 000 -8 000 Daltons)and flat width of 10 mm were utilized for dialysis which was purchased from spectrum labs.Dialysate used was distilled water to ensure successful removal of impurities to create hydrogels using the chitin and chitosan samples by modifying on protocols mentioned by Tamura et al［4］.

1.1 Preparation of chitin from squid pen samples

The squid pen samples were purified using distilled water in order to remove the various impurities and unwanted proteins that are naturally found in pens.Then squid pens were washed in a 4% NaOH bath at 80 -90 ℃for 1 h.Samples were washed with deionized water and treated with 1% HCl solution for 24 h.Then samples along with solutions were stirred for 20 min before removing samples and washing with deionized water to ensure demineralization.Chitin samples were subsequently placed in an air-dry oven at 50 ℃to cure for about 1 h.In order to promote the dissolution of chitin and chitosan samples they were first ground into a fine powder with an industrial grinder.

1.2 Dissolving chitin/chitosan

To prepare the dissolved chitin/chitosan，solvent system of 150 g of CaCl2·2H2O along with 150 mL of methanol was prepared and refluxed for 1 h below 90 ℃.And 10 g of(Humboldt and Loligo)chitin samples were dissolved in the solvent separately，also by refluxing for 30 min at 60 ℃.Both of these solutions were then subjected to dialysis using a Spectra/Por 1 ® Membrane with MWCO of 6 000 - 8 000 Daltons.About 100 mL of each sample were placed in the membrane，and each membrane was placed in 1 L of deionized water.The water was changed every 2 h for 24 h.

Both Loligo and Humboldt squid chitin samples were prepared by dissolving 1 g of chitin;(after grinding)in 100 mL of solvent with 90% acetic acid.Sample was left to stir overnight at 50 ℃.While Loligo squid chitosan sample was also prepared by dissolving 1 g of chitosan sample in 100 mL of solvent with 10% acetic acid，and another 1 g in 5% acetic acid.These solutions were also stirred overnight at 50 ℃.Later the samples were subjected to dialysis using Spectra/Por 1 ®membrane with MWCO of 6 000 -8 000 Daltons.

1.3 Viscosity measurement

Viscosity measurements of the chitin samples were taken using a Cannon Ubbelohde C71-100 viscometer.A Cannon Ubbelohde 1B-L459 viscometer was used to measure the chitosan solution.The sample solutions were first filtered through a Millipore filter (pore size:0.4 μm)in order to remove any undissolved chitin particulates that might cause the capillary to become narrow or clogged.And 10 mL of these filtered solutions were then transferred to the appropriate viscometer and readings were taken at different dilutions for each sample.These measurements were all taken at room temperature［5］.

1.4 pH measurement

The pH values of these solutions were all measured using a Thermo Scientific Orion Star*pH meter and electrode.The readings were taken after the pH meter indicated that it had finished equilibrating.These readings were taken at (21.1 ±0.06)℃.

1.5 FTIR measurement

FTIR readings were taken of 1 g of Loligo and Humbodlt squid chitin and Loligo chitosan.Chitin was grounded to powder before dissolving.Thermo electron FTIR with Nexus 470 bench and continuum microscope were utilized at College of Textiles，North Carolina State University，to perform qualitative analysis of chitin and chitosan samples in solid form.IR microscope was used to analyze powdered samples，which were compared with resolutions that have been predetermined and catalogued in SpectaTMsoftware for presence and identification of different amine group's presence in structures.Amine group's presence provides for antibacterial activity［6］.

2 Experimental

Humboldt and Loligo chitin samples were successfully dissolved in the calcium chloride (CaCl2)/methanol (CH3OH)solvent system at 60 ℃.Solutions obtained were fairly viscous and transparent.However，as soon as chitin solutions were brought to room temperature or 37 ℃，the samples turned opaque as if the chitin did not dissolve at these lower temperatures.In addition，when these chitin samples were dialyzed，it could still be seen that the samples were opaque.

Chitin/chitosan samples dissolved completely in acetic acid with small particulates which were found to be stable at 37 ℃.Solutions obtained were transparent and more viscous throughout the mixture.Measurements using the viscometer were utilized to calculate intrinsic viscosities for the solutions using the Huggin's and Kraemer's equations and method.The Huggin's method involves extrapolating the following equation to concentrations where it equals zero［7-8］:

where ηspindicates specific viscosity，which is calculated by dividing the difference between the efflux time at a certain concentration and the efflux time of the solvent.Concentration is noted as c，and the intrinsic viscosity is represented by［η］.kHis known as the Huggins constant and can be used to describe an ideal solvent.Kraemer equation for finding intrinsic viscosity is slightly different.The equation utilizes ηrrelative viscosity，which is calculated by dividing the efflux time at a certain concentration by the efflux time of the solvent［5，8］.

The Huggin's and Kraemer's equations utilize the linear relationship between the concentrations of dilute polymers and the viscosity of their solution.Intrinsic viscosities were found in both cases by extrapolating the equation to a concentration equal to zero［3，5］.

3 Results and Discussion

Chitin samples in CaCl2/CH3OH solvent remained undissolved at room temperature even after dialysis.Chitin solutions in CaCl2/CH3OH could not be characterized effectively using a capillary viscometer.The efflux time of the most concentrated solution was barely higher than the efflux time of the solvent alone.The data from the Humboldt and Lolgio chitin samples in acetic acid did not result in a plot that could be modeled effectively using the Huggins's and Kraemer's methods，seen in Figs.1 and 2.While Fig.3 shows chitosan in 5% acetic acid，results obtained from this plot are much closer to the ideal behavior of a dilute polymer in solution.Figures 1 and 2 show that according to the Huggin's and Kraemer's plots，the chitin samples viscosities are measured and extrapolated to zero，instead of being convergent lines，as the case of Fig.3 for the chitosan solution.The Huggin's constants reported in Table 1 for the chitin samples are negative，showing that solvent system utilized is not ideal.The Huggin's constant for the chitosan solution was 0.517，which was close to the range 0.2 -0.5 that showed solvent system used for chitosan and was clearly a good solvent.The sum of the Huggin's and Kraemer's constants (kH+kK)should be 0.5 for an ideal system.In Table 1，both chitin samples had negative values，whereas the chitosan sample was much closer to 0.5 at 0.431［7-8］.

Fig.1 Huggin's and Kraemer's plots for Humboldt chitin in 90% acetic acid

Fig.2 Huggin's and Kraemer's plots for Loligo chitin in 90% acetic acid

Fig.3 Huggin's and Kraemer's plots for Loligo chitosan in 5% acetic acid

Table 1 Intrinsic viscosities and Huggin's and Kraemer's constants for various samples

It was found that the viscosity decreased with increasing concentration of acetic acid.This might be attributed to a“polyelectrolyte effect”.This effect describes the phenomenon in which increasing the concentration of a polymer in an aqueous solution exponentially decreases its viscosity;this is apparent for both chitin samples in acetic acid［5］.Conversely，the chitosan sample in acetic acid behaved nicely without any“polyelectrolyte effect”which was hypothesized［9］.

In order to determine an average molecular weight，one can use the Mark-Houwink-Sakurada relationship， as shown below［8］.

where［η］reprensents intrinsic viscosity，Mvis the viscosity of average molecular weight，while a and K are constants that depend on the solvent system and polymer that can be determined experimentally.In the case of the chitin samples，the constants K and a in acetic acid could not be found.

Measurements of the pH obtained experimentally from the chitin/chitosan samples together with acetic acid showed that they had slightly greater acidic pH，compared with the acetic acid solutions alone，see Table 2.

Table 2 pH measurements of various samples at 21.1 ℃

In the case of the chitosan solution，the following constants were found for a similar solvent system，and were used for these experiments to calculate an approximate viscosity average molecular weight.The constants selected were for a 2%HAc/0.2 mol/L NaAc solution:K = 13.8 × 105and a = 0.85 reported previously［8］.This led to a viscosity average molecular weight of 3.9 × 10-7calculated using the Mark-Houwink-Sakurada equation［3，10］.

FTIR spectroscopy provided another method to examine the differences between the chitin samples as well as the chitosan sample.One can see the spectra from all three samples in Fig.4.As expected，the characteristic —OOH stretch band was present in all three samples between about 3 000 and 3 500 cm-1.The spectrum for the chitosan sample，however，also showed a more pronounced peak at 3 350 cm-1，which could be indicative of a stronger presence of primary amines.The samples also exhibited the characteristic absorption bands for amides at roughly 1 620 and 1 570 cm-1［6］.It is also noticeable that the relative size and proportion of these amide absorption bands are relatively lower when compared with the —OOH stretching for each sample.Overall，it would seem that the spectrum of the chitosan sample was similar to the two spectra obtained from the Humboldt and Loligo chitins.One of the most obvious differences between the chitosan and chitin spectra is the strong absorption band found in the chitosan spectrum at about 1 410 cm-1.This peak can be attributed to the CH3groups in the acetyl grouping，which is formed after deacetylation of the chitin to chitosan.Alternatively，these can be attributed to other amine moieties that may be present in these types of chitin.It is suggested that further study needs to be done to determine amine presence and peaks in squids［6］.

Fig.4 FTIR spectra of (a)Loligo squid chitosan，(b)Humbodlt chitin，and (c)Loligo chitin

4 Conclusions

The use of acetic acid as the solvent system that can be utilized and is close to an ideal solvent for these types of chitin.In comparison，the CaCl2/CH3OH solvent system did not provide as ideal solvent to dissolve the chitin or chitosan samples.Mild acetic acid with dilute water as a solvent helped in dissolving chitin，which that dilute acids could be utilized to dissolve chitin after removal of respective protein attached to it and associated with biological species from which it has been derived.Loligo squid chitin was found to have higher molecular weight as compared with Humboldt squid chitin.An acidic pH shift was reported with chitin and chitosan structures with the use of acetic acid.Chitin and chitosan structures are acidic in nature，which can be controlled by performing dialysis once dissolved in an aqueous solution.An adjustment of the pH and the FTIR spectroscopic analysis helped in revealing the presence of amines groups in both chitosan samples and the chitin samples，but stronger amine presence was revealed in case of chitosan which could be due to deacetylation associated.The presence of amine groups in chitin was negligible as compared with the Loligo squid chitosan sample.Further studies are currently being planned utilizing wounds international reports［11］，which suggests that silver can be utilized along with β-chitin structures that can develop preferred biomaterials for wound dressing's applications utilizing mild acidic solvents to dissolve.

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