Characterization and Physical Chemistry of Chitosan and Methoxypoly

Characterization and physical chemistry of chitosan and methoxypoly (ethylene glycol)-g-chitosan and its correlations with aqueous solubility

Introduction

Chitosan is a linear polysaccharide composed of a linear β-(1  4)-linked copolymers of 2-amino-2-deoxy-D-glucopyranose (GIcN) and 2-acetamido-2-deoxy-D-glucoopyranose (GIcNAc) units (figure 1). it is produced by N-deacetylation of chitin, an aminopolysaccharide commonly found in the exoskeletons of insects, crustaceans, and the cell wall of some fungi. It was claimed that chitosan is synthesized when the degree of deacetylation (DD) of chitin reaches about 50% (variable depending on starting polymer) and becomes soluble in aqueous acidic media (figure 1). Chitosan is water soluble in acidic media mainly because of the formation of polyelectrolyte in acidic media, by protonating the NH₂ function on the C-2 position of the D-glucosamine repeat unit. Chitosan catches a lot of attentions in science field over the decades because of its various applications of commercial and biomedical uses. Chitosan is the only pseudonatural cationic polymer. A lot of unique characters of chitosan are found and lead to many applications. The biomedical applications of chitosan are available to achieve based on its unique biological activities, such as low cytotoxicity, biodegradability, antimicrobial activity, and antioxidant capacity. However, some of the most important applications of chitosan are prohibited by its poor solubility in natural and alkaline media. As discussed above, the solubility of chitosan highly depends on its degree of deacetylation, more specifically, the protonation of NH₂ group in acidic media, pertaining to its GIcN units. The lack of acidic condition significantly decreases the solubility of chitosan in aqueous media, which makes many of its biomedical applications impossible. It was found that the degree of ionization of chitosan depends on the pH and the pK of the acid in the media. An average degree of ionization α of chitosan with low degree of deacetylation is around 0.5, which in HCl, α = 0.5 corresponds to a pH of 4.5-5, an absolutely acidic media. The distribution of the acetyl groups along the main chain may also contribute to the solubility of chitosan in aqueous media, as well as the total molecular weight.

Figure 1. synthesis of chitosan by N-deacetylation of chitin.

Several derivatizations have been proposed to improve the polymer properties of chitosan. Recent study shows a water-soluble form of chitosan at neutral pH was obtained in the presence of glycerol 2-phosphate, at pH 7-7.1 and room temperature. Furthermore, one of the most explored derivatives of chitosan is poly (ethylene glycol)-grafted chitosan due to its advantage of being water soluble in neutral and alkaline media, depending on its degree of grafting. Methoxypoly (ethylene glycol)-graft-chitosan (PEG-g-Ch), derivative of chitosan, displays tremendously improved solubility in aqueous media, with a large range of pH values from 1.0 to 11.0. PEG-g-Ch is synthesized by grafting non-ionic and highly hydrophilic poly(ethyleneglycol) chains on chitosan. Poly (ethyleneglycol), as a non-biodegradable polymer, provides promising potential to be a great substitution of chitosan for biomedical applications. One major concern regarding biomedical application is about toxicity of such derivatives, while literature showed the improved biocompatibility and in vivo bioavailability of PEG-g-Ch as compared to its parent chitosan. The study of solubility of a molecule often focuses on its physical and chemical properties because the solubility of a substance fundamentally depends on the physical and chemical properties of the solute and solvent. Physical properties and characterization of PEG-g-Ch, including different average degree of deacetylation, higher average of N-substitution and lower intrinsic viscosity, are believed to associate to its greater aqueous solubilization in neutral and alkaline media as compared to its parent chitosan. The average degree of deacetylation (DD) is an important physical property for chitosan, as it often determines whether the biopolymer is chitosan or its precursor, chitin, in which tells how the polymer can be applied. Such concept can also be applied to the study of water solubility of PEG-g-Ch and its parent chitosan by determining the correlation between average degree of deacetylation and its solubility. A simple and rapid method is thus required for determining the DD, including liquid and solid nuclear magnetic resonance (NMR), which is discussed in this paper. In general, PEG-g-Ch exhibits a much different degree of deacetylation, as compared to chitosan. In the reference paper, it was found that mPEG shows predominant N-selectivity. Although O-substitution was also reported, they generally call for the execution of multi-step reactions in the laboratory, while the improved water solubility is only attained at high average degree of substitution. PEG-g-Ch also displayed much lower intrinsic viscosity, as compared to the parent chitosans, showing the extensive exposition of PEG chains to the aqueous medium and compact coiling of the chitosan backbone. It was found that, the PEG chains grafted onto the chitosan also determined the crystalline arrangement, showing the thermal stability. In addition, it was interesting to notice that one PEG-g-Ch derivative showed a distinct rheological behavior as it formed a physically cross-linked hydrogel that exhibited a thermo-induced sol-gel transition at 38 C.

The aim of this paper is to investigate and study the characterization and physical chemistry of PEG-g-Ch and its parent chitosan, how their intrinsic viscosity and average degree of deacetylation contribute to the aqueous solubility. The rheological behavior of PEG-g-Ch derivatives is also discussed regarding its contribution to the solubilization. The study targets on the physical properties of PEG-g-Ch, the chitosan derivative, as a tool to improve the understanding on the structure property relationships between PEG-g-Ch and its parent chitosan to support its biomedical applications.

Degree of Deacetylation

The insolubility of chitin is a major problem that prevent the development of processing and uses of chitin. Although there are other chemical and physical properties associated with its solubility, the average degree of deacetylation (DD) of chitosan is essentially related to its aqueous solubility, as compared to chitin. This subtopic talks about the correlation between average degree of deacetylation and its water solubility. As mentioned in the introduction, a chitosan is said to be formed, when the average degree of N-deacetylation of chitin reaches about 50% in an acidic media. As a major structural difference between chitin and chitosan, in order to study its aqueous solubility, the characterization of a chitosan sample requires the determination of its average DD. Various methods have been proposed for the determination of DD, including IR, elemental analysis, UV, and NMR.

Figure 2. ¹H NMR spectrum of chitosan in D₂O, pH ~4.0, T = 85 C.

¹H NMR is considered as the most convenient technique for the measurement of the acetyl content of soluble samples. Figure 2 shows a ¹H NMR spectrum of chitosan dissolved in D₂O (pH ~4.0, T = 85 C). The signal at 1.95 ppm allows the determination of acetyl group of N-acetyl-glucosamine. The signal at 4.79 ppm determines the proton of glucosamine units, and the signal at 4.50 allows the determination of proton of N-acetyl-glucosamine. The integral intensity of these proton signals can be measured and be used to determine the average degree of deacetylation (DD). The DD of the sample chitosan can be determined by the formula below:

$\mathit{DD}=[1–(\frac{1/3I_{{\mathit{CH}}_3}}{1/6I_{H_2{–H}_6}}\left)\right]\times 100$

Where I_CH3 is the integral intensity of CH₃ and I_H2-I_H6 is the summation integral intensities of from H₂ to H₆. ¹⁵N solid NMR is also often used to study DD, giving two signals related to the amino group and the N-acetylated group. Figure 3 shows the ¹⁵N NMR spectra of (a) chitin, (b) homogeneous partially reacetylated chitosan, (c&d) heterogeneous commercial chitosans. The signal at 120 ppm in (a) and (b) allows the determination of N-acetylated group, and the signal at 15 ppm in (b), (c), and (d) shows the presence of amine group, providing direct evidence of N-deacetylation, which can be used to monitor the N-deacetylation of chitin.

Figure 3. ¹⁵N NMR spectra of (a) chitin, (b) homogeneous partially reacetylated chitosan, (c and d) heterogeneous commercial chitosans.

Table 1 illustrates the degree of acetylation of corresponding chitin and chitosans discussed above, obtained by ¹H NMR, ¹³C NMR and ¹⁵N NMR. Sample A (chitin) shows no deacetylation, since both DA acquired from ¹³C NMR and ¹⁵N NMR equal 1, meaning no N-deacetylation occurred. This explains the insolubility of sample A in the aqueous solvent system. On the other hand, sample B, C, and D (chitosan) show great deacetylation, with DA range from 0 to 0.6 accordingly. In addition, all three samples were dissolved in aqueous solvent under acidic condition (pH 4.0), showing its water solubility as compared to sample A.

Table 1. Degree of acetylation of chitin and chitosan.

This can conclude the water solubility of chitosan is correlated to its average degree of deacetylation, however, it is also noticed that chitosan is limited to aqueous media with low acidity, meaning study of solubility with variable pH values is necessary. Study showed the average degree of polymerization chitosan depends on both the average degree of deacetylation and on the ionic strength of the medium, meaning the solubility of chitosan is pH dependent. In fact, study showed that chitosan samples reached pKa 6.4 and pKa 7.3 when DD was extrapolated to 100% and 0%, respectively, meaning at pH around 7.0 the degree of dissociation of the deacetylated chitosan is very low. Thus, highly deacetylated chitosan sample (DD > 80.0%) can show low solubility in a neutral media, behaving almost entirely discharged, while chitosan sample with low deacetylation (DD < 10%) can contain significant amount of positive charges in acidic media, indicating the variable factors on the water solubility of chitosan. In fact, the solubility of chitosan is a very difficult parameter to control. It is strongly related to the DD, the ionic concentration, the pH, and even the distribution of the acetyl groups along the chain. Figure 4a shows the solubility of the chitosans and PEG-g-Ch derivatives evaluated by measuring the transmittance of the polymer solutions as function of pH. The solubility was estimated from the measurements of transmittance by using UV spectrophotometer. When high transmittance values were observed, it represented high solubility, while low transmittance was associated with low solubility. It illustrates the high solubility of PEG-g-Ch in the whole pH range, showing no significant connection between its solubility and ionic strength of the medium. Now the question remains in this case: would the average degree of acetylation also play a major role in its water solubility?

Figure 4. Water solubility as function of pH of chitosan and PEG-g-Ch samples (a); TG curves of Ch and PEG-g-Ch samples (b); thermos-stability of chitosan and PEG-g-Ch samples (c&d).

The answer is no, in fact, PEG-g-Ch shows very different N-acetylation. Table 2 provides values for average degree of deacetylation (DD) of chitosan and PEG-g-Ch samples. It shows, despite the great solubility in a large pH range (figure 4), PEG-g-Ch samples show significantly different DD, as compared to its parent chitosan. Such behavior was expected, since PEG-g-Ch is synthesized by grafting non-ionic and hydrophilic poly(ethyleneglycol) chains on chitosan, replacing the amine groups. In fact, all three PEG-g-Ch samples show lower DD than their parent chitosans. Considering the high solubility of PEG-g-Ch at large range of pH, it is hard to find the correlation between the average degree of deacetylation and its solubility. However, it is clear that grafted chitosan shows different average degree of deacetylation, showing different physical property, as compared to its parent chitosan.

Table 2. Values of average degree of deacetylation (DD) of chitosan and PEG-g-Ch samples.

Intrinsic viscosity

Another important characteristic of these polymers is the molecular weight and its distribution. Intrinsic viscosity measures the contribution of a solute to the viscosity of a solution. The study of intrinsic viscosity in polymer chemistry is extremely important, because it provides first-hand molecular characteristics of the polymers. In polymer chemistry, intrinsic viscosity is related to molar mass based on the Mark-Houwink equation. The molecular weight of polymer can be calculated from intrinsic viscosity using the Mark-Houwink relation, vice versa, with known values of the parameter K and a:

$\left[\eta \right]=K{M}^a$

Where both a and K depend on the particular polymer-solvent system, which can often be found from the literature. In size-exclusion chromatography, the intrinsic viscosity of a polymer is directly related to the elution volume of the polymer. Both a and K can be determined after running several samples of polymer. Table 3 illustrates the values of average degree of deacetylation, intrinsic viscosities of chitosan and PEG-g-Ch samples, and average degree of substitution for PEG-g-Ch samples. The intrinsic viscosities of samples were determined by using viscometer. In this study, the concentrations of the polymer solutions were all below the critical polymer concentration, assuring the experiments were done in dilute regime. However, amine groups on the chitosans can cause the electrostatic repulsion on the solution behavior of the polymers and must be considered in a diluted acid media. But in the case of chitosan samples in this study, the high ionic strength in the solution can overcome such electrostatic repulsion, proved by the linear behavior carried out by the curves η vs. concentration illustrating that the intrinsic viscosity values may be directly related to the viscosity average molecular weight of the polymers, thus the calculation was trustworthy. On the other hand, the electrostatic interaction was much less important in the case of PEG-g-Ch derivatives because of the occurrence of extensive N-graftization. The lower intrinsic viscosity of those derivatives strongly suggests the compact coiling due to the PEG chain preventing the intermolecular association. It was also found that, despite the different intrinsic viscosity, PEG-g-Ch samples showed similar degree of N-substitution (~40%). A conclusion can be made that the physical behavior of PEG-g-Ch derivatives in diluted aqueous solution is mainly determined by the PEG chains grafted onto its parent chitosans.

Table 3. the values of average degree of deacetylation, intrinsic viscosities of chitosan and PEG-g-Ch samples, and average degree of substitution for PEG-g-Ch samples.

The X-ray diffraction analysis also illustrates the high crystallinity of PEG-g-Ch derivatives due to the presence of numerous PEG chains grafted on the backbone. Figure 4b shows the XRD pattern of chitosan and PEG-g-Ch sample. According, the chitosan pattern shows low intensity peak, which is taken as evidence of its low crystallinity. In contrast, the XRD pattern for PEG-g-Ch shows significant higher intensity peaks, which are also found on the pattern of PEG-COOH. Such XRD results highly suggest the high crystallinity and compact coiling of PEG-g-Ch. Figure 4c and d shows the study of thermal stability of chitosan and PEG-g-Ch samples by TG analysis. The graphs reveal that there was great weight loss for chitosan in all three stages, while PEG-g-Ch derivatives showed no significant weight loss till stage three. The different behaviors may be attributed to the high arrangement of PEG grafted chitosans, precluding the absorption of water, showing the high thermal stability. Both the great crystallinity and high thermal stability can be related to its low intrinsic viscosity as discussed above.

Rheological Behavior

The study of rheological behaviors of chitosan and its derivatives was done in the concentrated environment. It was reported that physically cross-linked hydrogel were formed from highly concentrated aqueous solutions of PEG-g-Ch derivatives at neutral pH. The sol-gel transition was partially reversible, and the gelation temperature depended slightly upon experimental condition. Figure 5a reveals the dynamic viscosity of PEG-g-Ch derivatives decreased as the decrease of their shear rates, while figure 5b shows no dependence of dynamic viscosity on temperature. Taking into account the great difference of intrinsic viscosity between PChCm and pChD3 according to Table 3, the dynamic viscosity of the two derivatives revealed similar values, as compared to PChD1 and PChD2 with much lower dynamic viscosity. Such behaviors may be attributed to the content of GIcN units found on the chains of the corresponding polymers, relating to the short-range intermolecular associative forces. In the case of PChD3 and PChCm, the high content of GIcN units in the chains may favor the establishment of hydrogen bonds, provoking the expansion of the hydrophilic shell. The rheological behavior of such derivatives was affected by the more pronounced polymer coil interpenetration caused by the high content of amino groups in the chains of such derivatives, known as GIcN units. Figure 5c reveals that the storage modulus (G’) of sample PChCm, PChD1, and PChD3 are higher than their corresponding loss modulus (G’’). However, same behavior did not apply to PChD2. In contrast, PChD2 exhibited lower storage modulus (G’) than loss modulus (G’’) at 12.5 rad/s, claiming the formation of a structured hydrogel.

The similar behavior was found on PChCm and PChD1 on temperature sweep measurement, despite the significant difference on their parent chitosans, in terms of DD and intrinsic viscosity (table 3). The increase of G’ and G’’ in the range of 25-40 C was associated to the thermo-induced rearrangement of the polymer structure. The somehow constant G’ and G’’ values shown by PChD3 in this range indicated its different rigid network, which was slightly affected by the thermos-induced rearrangement. The sol-gel transition of PChD2 occurred at 38.2 C, which may be attributed to a particular combination of DD and intrinsic viscosity.

Figure 5. Shear flow curves (a), dynamic viscosity vs. temperature (b), frequency sweep measurement (c), and temperature sweep measurement (d) of PEG-g-Ch derivatives.

It was found that the rheological behavior of PEG-g-Ch derivatives is mainly affected on the subunits of its chitosan chains, while presenting approximately the same average degree of substitution. Both frequency sweep and temperature sweep measurement revealed that the gelation of PChD2 was directly related to its average degree of deacetylation of intrinsic viscosity. Such phenomenon provides a promising application of drug delivery platform in the future.

Conclusion

The graft of methoxypoly (ethylene glycol) onto chitosan led to the production of N-substitution chitosan derivative, revealing fully aqueous solubility and large range of biomedical applications. PEG-g-Ch derivative shows different average degree of deacetylation, as compared to its parent chitosan, but its ionizable functional groups lead to fully aqueous solubility, covering large pH range. Due to its high degree of N-substitution, which consumes its original amine groups responsible for their polyelectrolyte behaviors in chitosan, to large molecular weight PEG chains, resulting in an accentuated conformational effect, the PEG-g-Ch derivative showed a much lower intrinsic viscosity, as compared to its parent chitosan. Despite the potential of other factors on the low intrinsic viscosity of PEG-g-Ch, the low intrinsic viscosity of this derivative is mainly contributed by its high molecular weight hydrophilic PEG chains by forming compact coiling structure. The grafted PEG chains also strongly affected the arrangement and the thermo-stability of the PEG-g-Ch derivatives, as revealed from the results of X-ray pattern and TG analysis. The similarity of X-ray pattern between PEG and PEG-g-CH indicated the crystalline arrangement in the solid state and the thermal behavior of such chitosan derivatives. The study of rheological behavior of the PEG-g-Ch derivatives reveals the influence from both parent chitosan and physical properties of its derivative, including its average degree of deacetylation and intrinsic viscosity. In particular, PChD2 resulted in a unique rheological behavior, showing a transition to solid-like behavior. A sol-gel transition at ~38.2 C was observed on PChD2, providing the possibility for the development of drug delivery platforms.

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