Low molecular weight chitosan (>78%) was purchased from Sigma Aldrich (St. Louis, MO, USA); tripolyphosphate (TPP), 4-formylphenyl boronic acid and sodium borohydride from Thermo Fischer Scientific (Bridgewater, NJ, USA); acetic acid, methanol, acetonitrile and glucose were purchased from Merck (Whitehouse, NJ, USA). All other chemicals were of reagent grade.

Four hundred micrograms of chitosan dissolved in 1% acetic acid solution was made to react with various quantities of 4-formylphenylboronicacid dissolved in methanol in the presence of sodium borohydride as reducing agent. The reaction was maintained at room temperature and made to run for 24 h. The resulting PBA-bonded chitosan conjugates (labeled as F1 through F6, Table 1) were separated from the reaction mixture by centrifugation and washing with methanol and then ethanol.

IR spectra of chitosan, TPP, chitosan- phenylboronic acid conjugate were obtained using a Perkin Elmer FTIR Spectrometer (Spectrum RX 1). Lyophilized samples were gently mixed with about 250 mg of micronized KBr powder that was oven-dried at 120 °C for 4 h. The mixture was compressed into a disc at 5000 psi for 5 min using a pneumatic press and infra-red spectra collected in the range of 2000–400 cm⁻¹.

The tagging of PBA onto chitosan was further ascertained using a time-of-flight secondary ion mass spectrometer (IV ION-TOF equipped with a liquid metal, Bi ion gun (LMIG), (IonTof GmbH, Munster, Germany), where boron ion (B⁻) was monitored as the secondary ion fragment from the boronic acid moiety of the conjugates.

Differential scanning thermograms were obtained using a Mettler Tolledo DSC system. The sample pan comprised of about 5 mg of lyophilized powder whereas the reference pan was a standard aluminum pan. Both sample and reference pans were heated from 25 °C to 500 °C at a heating rate of 10 °C/min under 20 mL/min purge of nitrogen gas. All samples were run in triplicate.

After a 1:10 dilution with deionized water, a drop of freshly prepared conjugate dispersion was placed onto an SEM imaging stub and left to air-dry at room temperature. The dried conjugate was then viewed under field emission-SEM (Model Quanta 400F, FEI Company, USA) at 3 kV.

Twenty micrograms of conjugate was added to 1 mL of 2 mg/mL glucose solution buffered at pH 5.4. The mixture was occasionally shaken for 1 h after which 50 μL of the supernatant was aspirated and reacted with a hexokinase kit (POINTE Scientific INC). The amount of glucose adsorbed onto the conjugates was determined as reduced nicotidamide adenine dinucleotide (NADH) by HPLC after subtraction of the unreacted glucose from the original.

A summary of the schematic chemical reaction between 4FPBA and chitosan leading to the formation of the conjugates is as shown below:

FTIR spectra of chitosan and conjugates (Figure 1) showed the characteristic peak for chitosan at 1655 cm⁻¹ attributable to an amide-linked carbonyl group, whilst the peak at 1325 cm⁻¹ corresponds to a secondary amide, most likely due to incomplete de-acetylation of chitosan [25,26]. The peak relating to the primary amine of chitosan at 1590 cm⁻¹ [27] became less apparent from F1 through to F6 and has virtually disappeared in conjugate F6. This strongly suggests that the PBA resides at the primary amine group (–NH₂). The bonding of PBA on the conjugates is confirmed by the presence of peaks at 1560, 1515, 830 and 630 cm⁻¹ which indicates the formation of p-di-substituted benzene [28]. This can only be explained if the PBA has been covalently attached to chitosan, as no such structure exists in “naked” chitosan.

The FTIR data for F1 and F2 shown in Figure 1 suggest a non-existent PBA functionality, i.e., at lower 4-FPBA loading, since the peaks representing the p-di-substituted benzene are not observed here. The ToF-SIMS analysis, however (Figure 2), revealed the presence of boron ions (B⁻) at 11.01 m/z in F1, F3 and F5, (i.e., representation of all the formulations studied). Furthermore, the intensities of these B⁻ peaks increase in proportion to the 4-FPBA load (Table 1). The B⁻ comes from tagged PBA of the conjugate since it does not appear in the pure ToF-SIMS spectrum. Thus, the ToF-SIMS analyses is in concert with the FTIR analysis in that there is a direct correlation between the degree of PBA bonding onto chitosan and the 4-FPBA loading during reaction, albeit the ToF-SIMS is more sensitive than the FTIR analysis and hence able to detect PBA functionality at lower concentration of bonded PBA within the formulations.

BA reversibly reacts with cyclic diols functionalities such as glucose to form 5- or 6-membered cyclic complexes [3]. The interaction between BA and diols may depend on the pKa of the BA and that of the diol, the pH of the medium and the nature of the buffer and solvent used [4]. Unfortunately, the interrelationships between these factors and the propensity for glucose to adsorb are poorly understood. The present study was aimed at correlating the changes in physicochemical properties of the formulated conjugates with their sensitivity to glucose adsorption (2 mg/mL). The data in Figure 3 shows that there is a direct correlation between the amount of bonded BA on the conjugates with the glucose adsorption sensitivity of F1, F2 and F3. Conversely, the sensitivity to glucose adsorption on F4, F5 and F6 reduces in that order at glucose concentration studied (F3 adsorbing the highest amount of glucose in all the formulations).

SEM images of the conjugates presented in Figure 4 reveal a porous matrix for F1, F2 and F3 whilst those of F4, F5 and F6 appear to be crystalline. It seems plausible from the SEM images of the conjugates that due to the porous nature of F1, F2 and F3 matrices, glucose adsorption onto these conjugates proceeds unrestricted. In contrast, due to the crystalline matrices of F4, F5 and F6, adsorption by glucose is inhibited because of a close packing arrangement even though these series (F4, F5 and F6) have a higher concentration of bonded PBA compared to the former series. Nevertheless, the glucose adsorption study corroborates the FTIR/ToF-SIMS analyses for F1, F2 and F3 in that a positive correlation exists between glucose adsorption and the concentration of PBA bonded onto these conjugates.

In a related study Wu et al. [24] attributed restricted swelling of PBA-chitosan nanoparticles (an indication of poor PBA-glucose interaction) at high PBA to chitosan concentration to the bidentate binding nature of glucose by two the two –OH groups within the BA moiety. In this regard, the adsorbed glucose anchors as a cross-linker and hence restricts swelling of the PBA-chitosan nanoparticles which impedes further glucose adsorption due to inaccessibility of glucose to further adsorption by PBA. This effect of reduced PBA-chitosan swelling due to adsorbed glucose was enhanced when the concentration of PBA was high. In the present study, the SEM investigations point to the formation of crystalline matrices in F4, F5 and F6, (i.e., at higher PBA-chitosan concentration). It appears that the change from porous (F1, F2, F3) to crystalline conformities accounts for the decreased sensitivity to glucose adsorption.

To study the change in material organisation in chitosan due to bonding with PBA, DSC analyses of the conjugates were performed and are presented in Figure 5 as (a) F2, F3 & pure chitosan, (b) F4, (c) pure chitosan. Due to their facile dehydration on heating, pure PBA crystals tend to give inconsistent melting temperatures; thus, true melting points are reflective of decomposition temperatures [29]. In all of the thermograms, an endothermic peak was manifested at 86.4 °C and is due to the liberation of water from the –NH₂ and –OH groups within the chitosan backbone [30]. An exothermic peak from pure chitosan (Figure 5c) occurs at 308.3 °C and represents decomposition of –NH₂ groups. This decomposition of –NH₂ groups also occurs in conjugates F2, F3 and F4 (Figure 5a,c), albeit at a lower temperature (293.2 °C). A reduction in decomposition temperature of chitosan is due to consumption of –NH₂ groups by bonded the moiety [31] and in the present study, attests to the bonding of PBA to chitosan.

All three conjugates registered exothermic peaks at 338.5 °C which is absent in the pure chitosan thermogram (Figure 5c), therefore we can ascribe this exothermic peak to bonded PBA, which interestingly, increases in intensity with increases in PBA to chitosan concentration. We can conclude that the exothermic peak at 338.5 °C is related to the melting enthalpy of the bonded PBA moiety within the conjugates which is also responsible for imparting crystallinity to the conjugates. This conclusion is supported by a similar observation with chitosan-poly vinyl alcohol (PVA) conjugates where it was reported that the degree of crystallinity of the conjugates decreased with increase in chitosan content, given that the crystallinity of the conjugates was due to PVA [32].