3‐1 Hydrogel formation
3‐1‐1 Prepared of stabile CHC/SAL combination solutions
To the authors’ knowledge, there has to date been no reported method to provide a steady combination solution with chitosan and sodium alginate. This owing to two main reasons: (1) the opposite charge of the polymers which leads to electrostatic attraction promoting aggregation8; (2) the property of sodium alginate forming an acid gel by the addition of acidic chitosan solution3. This study focused on the compatibility and stability of the combination solution for realistic applications and characterization of a calcium chloride induced gelation of the CHC/SAL combination solution, with the resulting composite gel being characterized with regard to dynamic mechanical properties and potential as a drug delivery platform.
Ordinarily, an acidic chitosan solution would rapidly produce a hydrated precipitate upon addition of a strong base, such as NaOH. This due to the reduced positive charge density along the chitosan chains (NH3+
). The neutral chains would interact strongly through hydrogen bonding and hydrophobic interactions between chains. In this study, amphiphilic carboxymethyl‐hexanoyl chitosan (CHC) was used because of its proven potential as a hydrophobic drug carrier, utilizing self‐assembly6. To prevent the phenomenon of aggregation and acid gel formation, the amphiphilic CHC solution was adjusted to slightly
alkaline (pH=7.5~8) before combination with the sodium alginate solution. There was no hydrated precipitate but only a little agglomerate when the amphiphilic CHC solution was adjusted to weak base upon addition NaOH. This because of the steric effect from the long hexanoyl groups prevents hydrogen bonding between CHC chains48 and because the self‐assembly property into a micelle structure may further reduce the aggregation tendency.
At this pH value, the zeta potential of the CHC solution would be close to neutral, because the isoelectric point (IEP) was determined to be about 7.5 in previous study6. The fact that the modified chitosan is neutrally charged, and soluble at slightly alkaline pH, allows for the successful combination with sodium alginate. Subsequently, hydrogels can be formed by exposing the combination solution to gelation medium (calcium chloride solution in this study).
3‐1‐2 Formation of CHC/SAL composite hydrogels
SAL is well known to form a strong gel upon exposure to calcium ions. The calcium cross‐links the alginate chains by replacing sodium ions with calcium ions to form the well‐known “egg box” structure by electrostatic force between guluronic groups expressed on different alginate chains and bridging calcium ions. Thus, the gelation process was mainly driven by electrostatic attractions (ionic bonding) between Ca2+ ions and the SAL matrix.
However, several other types of interactions, such as hydrogen bonding, electrostatic repulsion, and hydrophobic interactions should also be present in the gel system. The hydrophilic carboxymethyl and hydrophobic hexanoyl substitutions of CHC induces self‐assemble into micelles having diameters in the range 50‐200 nm when the CHC concentration is above the critical micelle concentration (CMC), as is the case in the combination solution. Therefore, the structure of the formed composite gels is a crosslinked alginate matrix with embedded nano micelles. Scheme 3‐1 shows the structure of amphiphilic CHC and sodium alginate, as well as schematic drawing of the formed composite gels.
Scheme 3‐1 Molecular structure of modified amphiphilic chitosan (CHC) and sodium alginate (SAL) and the suggested crosslink network after gelation by CaCl2 rich medium.
3‐1‐3 Gelation time measurement
The gel formation process and the gelation rate of composite hydrogels were observed at room temperature. The gelation process would firstly occur at the junction zone between the gel solution and gelation medium. The gelation time could be adjusted by changing sodium alginate and calcium chloride concentration, with the fastest gelation time being close to instantaneous and the longest being about 10 s (Table 3‐1). As an example, the gel with the weight ratios CHC/SAL/glycerol = 1/1/5, prepared by exposure to 1 wt% solution formed a gel in roughly 10 s. By increasing the SAL concentration from 1 to 2 wt%, keeping the other conditions constant, the gelation time displayed an obvious decrease from 10 to 3s.
With increasing ratios of Ca2+ in the gelation media, the gelation time was greatly decreased.
In fact, the gelation appeared to occur almost immediately for all but the samples with the lowest SAL concentration.
The investigated gels showed a close to immediate gelation under a high enough calcium concentration in the gelation media. The gelation process should follow a two‐step process: (1) Ca2+ transport by diffusion to the carboxylic sites in alginate chains; (2) the reaction between carboxylic groups and Ca2+ to form the egg‐boxing structure in junction zones25. The diffusion of calcium in the gel should scale with viscosity and concentration gradient of calcium ions, but the viscosity should not differ much between 1, 1.5, and 2 wt%
SAL. Furthermore, the increase concentration actually increases both diffusion rate and the
amount of binding sites a Ca2+ meet. However, the most important reason is that the gelling that using the vial tilting method may actually measure gelation at the surface. As a result, the measurement of gelation time may attribute to the gelation process on the solution surface which making the gel become rigid and immovable.
1% SAL 1.5% SAL 2% SAL
1% Ca2+ 10 5 3
2% Ca2+ 3 less than 1 less than 1
3% Ca2+ 1 less than 1 less than 1
Unit: second(s) Table 3‐1 Gelation time of various condition hydrogels
3‐2 Equilibrium swelling degree under various swelling medium
The equilibrium swelling degree (ESD) of lyophilized composite hydrogels with the weight ratios CHC/SAL/glycerol = 1/1.5/0, prepared using gelation media with different CaCl2 concentrations, was determined in di‐water, cell culture medium (α‐MEM+10% FBS), and SBF (Fig. 3‐1). It was found that for all investigated swelling media, the ESD decreased with increasing concentration of CaCl2 in the gelation medium. In the case of di‐water, the ESD was 42.7 g/g for 1 wt% CaCl2 in the gelation media, while it was 31.2 g/g for 2 wt% CaCl2 and 26.9 g/g for 3 wt% CaCl2. The gels exhibited lowers swelling in the cell culture medium, and a minimal swelling in SBF.
For gels used in biomedical applications, the swelling is an important material parameter which greatly influences the substance exchange behavior, i.e. drug release. The extent to which a gel swells determined by the swelling pressure (π) of the gel. The swelling
pressure can be written:
π (3‐1)
where is osmotic pressure from the dissolution of polymer chains, is the osmotic pressure derived from counterions within the gel and is the elastic pressure derived from the deformation of the polymer network during swelling49‐51.
The term in the above equation is determined by the crosslinking density of the gels, where a high degree of crosslinking corresponds to a high elastic pressure opposing
swelling. The swelling property of a gel is thus closely related to the storage modulus of a gel.
In general, as crosslinking increase the storage modulus of the gel increases, while swelling capacity decrease. This leads to that a balance between the desirable macroscopic mechanical properties and swelling capacity has to be found. The hydrogels in this study being prepared using gelation media with different Ca2+ concentrations showed obvious differences in their swelling capacities. Compared with gels formed using gelation media with a CaCl2 concentration of 1 wt% the swelling decreased 27 % and 37 % for gels prepared using gelation media with 2 and 3 wt% CaCl2, respectively. In addition, the solvent was found to affect the swelling, as expected. As seen in Fig. 3‐1, the ESD was two times larger for samples swollen in di‐water than in α‐MEM, and the difference was even larger for sample swollen in SBF. The observed values of ESD can be explained by the compositions of the different swelling media. In deionized water the contributions of counterions to the swelling is the highest, i.e. is large. In contrast for α‐MEM and SBF the ionic strength of the swelling media is higher and the difference in ion concentration within the gel and in the swelling media is reduced, i.e. decreases. However, given the compositions of α‐MEM medium and SBF, their ionic strengths should be close to identical, and a similar swelling would be expected. One plausible explanation to the lower swelling for samples swollen in SBF is that SBF has a higher content of divalent ions, which are known to act as crosslinkers in alginate3. Such ions would increase the opposing elastic pressure in the above equation, leading to
reduced swelling. Calcium ions replacing sodium ions within the gel would also reduce the number of conterions within the gel due to their divalent charge. This phenomenon with polyvalent ions greatly reducing swelling of oppositely charged polymer gels is well described by Katchalsky52.
Fig. 3‐1 Equilibrium swelling degree of CHC/SAL composite hydrogels (weight ratio CHC/SAL/glycerol = 1/1.5/0) as a function of CaCl2 concentration in gelation medium. Gels were submerged in di‐water, medium (α‐MEM) or SBF for 2 days to reach the equilibrium state. Values reported are an average of n = 3.
1% CaCl2 2% CaCl2 3% CaCl2
3‐3 Water retention test for swollen CHC/SAL composite hydrogels
The ability to hold sufficient amounts of water inside the network structure is an important parameter to characterize hydrogels. As seen in Fig. 3‐2, all of the investigated hydrogels retained more than 80% of the absorbed water after 1 day at 25℃ and 54 % relative humidity. The composite hydrogels with weight ratios CHC/SAL/glycerol = 1/1.5/0 prepared using gelation medium with 1 wt% CaCl2 exhibited the highest water retention capability, the preparations utilizing higher concentrations of CaCl2 in the gelation medium (2 and 3 wt%) displayed very similar water retention behavior, i.e. the evaporation of water was the same.
Water retention tests performed under constant surface area, temperature, and relative humidity, revealed that the relative retention was also affected by the crosslinking through Ca2+ ions. Samples prepared using gelation medium with higher calcium content, i.e. higher crosslink density, exhibited a lower relative water retention capability. Increasing crosslinking density would decrease the ability to hold water inside the gel, as seen from swelling pressure equation (Eq. 3‐1). The swelling pressure of a gel at given water content could in theory be converted into water activity, which should influence the evaporation rate directly53. However, the prediction of how the relative water retention should vary with crosslink density is not trivial. The swelling pressure equation (Eq. 3‐1) is based on the simplified theories of Flory and Huggins54 and neglects many of influencing factors present in
our systems, such as phase behavior and gel structure. Both which should have major impact 25℃ and 54 % relative humidity. The samples had the weight ratios CHC/SAL/glycerol = 1/1.5/0 and were prepared using different concentrations of CaCl2 in the gelation medium.
Values reported are an average of n = 3.