Synthesis and characterization of chitosan-based pH-sensitive biofilm: An experimental design approach for release of vitamin B12

Ali Onal1, Hulya Demir1*, Olcay Kaplan Ince2, Muharrem Ince3

1Department of Nutrition and Dietetics, Yeditepe University, Istanbul, Turkey

2Department of Chemical Technologies, Munzur University, Tunceli, Turkey

3Department of Gastronomy and Culinary Arts, Munzur University, Tunceli, Turkey


Hydrogels because of their unique potentials, such as highwater content and hydrophilicity, are of interest for the controlled release of drug molecules. In the present study, a biofilm was produced using chitosan, a natural polymer. Characterization analyses of the synthesized biofilm were performed using Fourier-transform infrared spectroscopy (FT-IR), thermogravimetric analysis-differential thermal analysis, x-ray diffraction, and scanning electron microscopy-energy dispersive x-ray spectrometry. The analysis results of the film before loading were compared to the analysis made after adding vitamin B12 to the film. The release percentage of vitamin B12 was investigated by using ultraviolet-visible spectrophotometery. The FT-IR bands of vitamin B12 were in the range of 2,800-3,500 cm-1 and 1,000 cm-1-1,750 cm-1. When pH increased from 3 to 7, release of vitamin B12 from the biopolymer increased up to pH 5. While examining the release potential, parameters, such as pH, initial concentration of vitamin B12, release time, and solution volume, were optimized by using response surface methodology. It was determined that with the increase in release time and the initial concentration, pH contributed positively to the release of vitamin B12. The following conditions were determined for release of vitamin B12 from the biofilm: pH: 4.2, initial concentration of vitamin B12: 54.0 mg L-1, the release time: 117 min, and volume of solution: 2.1 mL. The analysis of variance results showed that the determination coefficients for the use of synthesized biofilm in vitamin B12 release were high and R2 =0.9704.

Key words: biopolymer, characterization, response surface methodology, vitamin B12

*Corresponding Author: Hulya Demir, Department of Nutrition and Dietetics, Yeditepe University, Istanbul, Turkey. Email: [email protected]

Received: 9 November 2023; Accepted: 6 March 2024; Published: 3 April 2024

DOI: 10.15586/ijfs.v36i2.2472

© 2024 Codon Publications
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0). License (


Hydrogels are three-dimensional, hydrophilic, and polymeric network structures that can absorb large amounts of water or biological fluids (Ayhan et al., 2007). Hydrogels are prepared by the polymerization reaction of one or more monomers and are insoluble because of the presence of chemical or physical cross-links between the main chains. Hydrogels have a wide usage, especially in medicine and pharmacy (Bajpai and Sharma, 2004). They resemble living tissues more than other synthetic biomaterials. This is due to the high water content and soft structure of hydrogels. Owing to the high biocompatibility of hydrogels, they are used in contact lenses, biosensor membranes, artificial skin material, and drug delivery systems. Hydrogels are classified according to their ionic charge, such as neutral, anionic, cationic, or ampholytic. Hydrogels exhibit swelling behaviour that changes depending on the external environment conditions. These polymers are called “physiologically responsive hydrogels” (Gupta et al., 2020). Main factors that affect the swelling behavior of physiologically or environmentally sensitive hydrogels are pH, temperature, ionic strength, and electromagnetic radiation. All pH-sensitive polymers contain acidic groups (e.g., carboxylic acid and sulfonic acid) and basic groups (e.g., sulfonic acids) that are capable of either accepting or donating protons in response to changes in environmental pH (Jin et al., 2016). Changes in environmental pH lead to structural changes, as ionizable groups are attached to the polymer structure, and there is a change in the dissolution of polymers and swelling behaviour of hydrogels. The structures of ionic polymers, which exhibit pH-sensitive behavior and are frequently the subject of study are poly(methacrylic acid) (PMAA), poly(acrylamide) (PAAm), poly(acrylic acid) (PAA), Poly(diethylaminoethyl methacrylate) (PDEAEMA), and poly(N,N’dimethylaminoethyl methacrylate) (PDMAEMA). However, polymers containing phosphoric acid derivatives were also examined (Ghorbanloo and Heidary, 2017).

Upon exposure to the aqueous medium of appropriate pH and ionic strength, some groups ionize and develop fixed charges in the polymer network, and pH-dependent swelling or shrinkage of the hydrogel causes electrostatic repulsive forces, resulting in drug release (Ghorbanloo and Heydari, 2017).

For any drugs to be administered orally, some physiological aspects of the body, and more specifically that of the gastrointestinal tract (GIT) must be recognized. For example, pH change occurs throughout the gastrointestinal system (Bajpai and Sharma, 2004); however, more delicate changes occur in various body tissues. Various approaches are used for colon-targeted drug delivery, including pH-dependent swelling-controlled systems (Bajpai and Sharma, 2004), delayed-release delivery systems (Gupta et al., 2020), intestinal pressure-controlled colon delivery capsules (Jin et al., 2009), and enzymatically degradable systems using a variety of enzymes (Ghorbanloo and Heidary, 2017).

A comprehensive review of the literature available so far on drug release studies has revealed that most in vitro release studies involved measuring of release rates from the drug-loaded polymer matrix at different pH environments. In order words, HCl solution was used to simulate gastric juice, and phosphate buffer with pH 6.8 was used to simulate intestinal fluid. Oral administration of therapeutic compounds is the most convenient, mode of administration, although it is not used for many drugs that are not able to overcome their many natural barriers (Dengre et al., 2000). The presence of pathological conditions affecting GIT also inhibits the absorption of active ingredients after oral administration. Vitamin B12 is an example of such drugs. Deficiency of vitamin B12 causes various diseases. Controlled release of vitamin B12 increases its concentration in the liver and blood serum level, and helps to prevent or treat cobalt deficiency in the body. Vitamin B12 has been preferred as a model drug because of its neutral behaviour and high solubility in water. Patients with pernicious anaemia and other intestinal disorders cannot absorb even a small amount of vitamin B12 found in food, causing vitamin B12 deficiency, therefore leading to lifelong intake of intramuscular injections of vitamin B12 (Bajpai and Sharma, 2004; Sarti et al., 2012). Adequate oral absorption of vitamin B12 is achieved by using its high daily doses (Gupta et al., 2020).

To date, no serious efforts have been made to increase the oral bioavailability of vitamin B12 by improving its penetration into the intestinal mucosa. Poly(N-vinyl-2-pyrrolidone) (PVP) is known as a good biocompatible compound. The non-toxic nature and higher hydrophilicity of PVP encourages its use for drug delivery devices. Another important point is that the synthesis of a gel does not require a high temperature or the use of some organic solvents, so the probability of loss of activity during the incorporation of a drug is minimal. The present study aims to close this gap in the field by regulating swelling behavior with the help of a cross-linker, designing drug delivery systems that can deliver the desired amount of vitamin B12 for the desired time, and with the experimental data to be obtained.

Polyacrylic acid (PAA) and its copolymers, because of their multifunctional structure, unique properties, and good biocompatibility, are used as carriers in drug delivery systems (Jin et al., 2009). Inter-polymer complexes between PVP and PAA are examined to develop new mucoadhesive drug carriers by taking advantage of hydrogen bonding between the carboxyl groups of both PAA and PVP. The adhesion strength and drug release rate of PVP-PAA inter-polymer complexes are controlled by varying the pH values as well as the mole ratios of PVP and PAA. The complex appears as an adequate carrier for the mucoadhesive drug delivery system (Jin et al., 2016). The copolymer can form both intrapolymer and interpolymer complexes between PVP and PAA components. PAA-cysteine (PAA-cys) conjugate has been shown to provide a relatively higher absorption of substrates in the rat intestinal mucosa both in vitro and in vivo (Ghorbanloo and Heidary, 2017; Jin et al., 2009). In addition, PAA-cys solution and microparticles have been recently highlighted in in vitro studies as having a promising potential for intestinal permeability enhancer of vitamin B12 (Ghorbanloo and Heidary, 2017).

The present study aims to synthesize and characterize various pH-sensitive polymers, to examine the release of vitamin B12 by loading into the synthesized polymers and to develop hydrogels suitable for vitamin B12 release.

Material and Methods

High-purity chemicals, including acrylic acid, chitosan, N, N`-methylene bisacrylamide, acetic acid, ammonium persulfate and vitamin B12 were purchased from Sigma Aldrich (ABD).

Use of Central Composite Design (CCD) in Experimental Studies

A CCD, which is a statistical approach, combined with the response surface methodology (RSM), is a suitable system for optimizing the levels of parameters that affect release of drug in the experimental study. This method is a useful method for analyzing the effects of some independent variables on RSM along with a set of statistical and mathematical data. In addition, various estimations and tests, such as estimation and testing of the regression equation with the CCD technique, the factor effect testing, model determination coefficient calculation, optimal level combination estimation, and response surface properties around it, are provided. The CCD is known as a regression analysis and is used to estimate parameters and establish relationships between test indices and periodic variables. The experimental design approach is particularly suitable for optimizing complex synergies or antagonistic effects between variable states (Ince and Ince, 2019). In this study, a CCD containing four and five levels of each of these factors was chosen to examine and optimize the effect of the selected variables. A CCD experiment design (Table 1) was created to measure the effect of each variable, namely pH (X1), initial concentration (X2), release time (X3), and solution volume (X4), on drug release. Variable levels were coded as -1, 0, and +1, respectively, and were defined as low, medium, and high variables. Also, +2 and -2 star points were defined for each experimental group, corresponding to +α and -α, respectively. The effects of main, interaction, and quadratic variables were modelled using CCD. This experimental design was carried out to minimize the effects of uncontrollable parameters and was based on controllable factors. In addition, for optimizing the process parameters, their interactions were performed using a CCD with a minimum number of experiments. The CCD design in question included the ranges of the independent variables that were preferred in the experimental study and that had an effect on adsorption, and the responses corresponding to these variables (Table 2).

Table 1. The analysis of variance (ANOVA) results of response surface quadratic model.

Source Sum of squares Df Mean square F-value p-value Prob > F
Model 6482.93 14 463.07 35.08 < 0.0001 significant
X1:pH 57.20 1 57.20 4.33 0.0549
X2: Initial concentration 226.63 1 226.63 17.17 0.0009
X3:Release time(min) 1,155.79 1 1,155.79 87.55 < 0.0001
X4:Solution volume 2,596.88 1 2,596.88 196.70 < 0.0001
X1X2 20.36 1 20.36 1.54 0.2333
X1X3 6.06 1 6.06 0.46 0.5083
X1X4 22.68 1 22.68 1.72 0.2097
X2X3 19.91 1 19.91 1.51 0.2383
X2X4 45.39 1 45.39 3.44 0.0835
X3X4 112.10 1 112.10 8.49 0.0107
X12 959.68 1 959.68 72.69 < 0.0001
X22 34.11 1 34.11 2.58 0.1288
X32 115.56 1 115.56 8.75 0.0098
X42 1,549.08 1 1,549.08 117.34 < 0.0001
Residual 198.03 15 13.20
Lack of fit 176.42 10 17.64 4.08 0.0670 not significant
Purity Error 21.61 5 4.32
Cor Total 6680.96
R2 0.9704
Adj R2 0.9427 29
Pred R2 0.8432
Adeq precision 22.004

Table 2. Independent variables and their levels.

Run Factor 1 Factor 2 Factor 3 Factor 4 Response
X1: pH X2: Initial concentration
X3: Release time
X4: Solution volume
Release (%) of vitamin B12(mgL–1)
1 3 50 60 1 49.8
2 3 80 120 1 80.8
3 7 50 120 1 78.4
4 7 80 120 1 92.2
5 7 50 60 2.5 78.4
6 3 50 120 1 73.1
7 5 65 30 1.75 78.2
8 3 80 60 1 66.0
9 7 80 120 2.5 99.0
10 5 65 90 1.75 100.0
11 7 80 60 2.5 93.8
12 7 50 60 1 58.8
13 5 65 90 1.75 95.0
14 5 65 90 1.75 98.0
15 9 65 90 1.75 74.2
16 3 50 120 2.5 95.1
17 5 95 90 1.75 95.0
18 5 65 90 1.75 99.0
19 3 80 60 2.5 85.7
20 5 65 90 1.75 95.0
21 5 65 90 0.25 44.4
22 7 80 60 1 67.2
23 5 65 90 1.75 96.7
24 3 50 60 2.5 84.2
25 3 80 120 2.5 93.3
26 7 50 120 2.5 95.0
27 1 65 90 1.75 73.1
28 5 65 150 1.75 100.0
29 5 65 90 3.25 90.1
30 5 35 90 1.75 90.7

Synthesis of Biopolymer Material and Loading of Vitamin B12

Biopolymer production

Acrylic acid, 10 mL, of was taken and filled with ultrapure water to make the final volume up to 50 mL. The solution was mixed in a magnetic heater stirrer until it became homogeneous. Then 0.5 g of chitosan was added, and mixing process was continued for 15 minutes. Mixing was continued by adding 0.6 g of N,N`-methylene bisacrylamide. Then 0.3 g ammonium persulfate (APS) was added and mixed.

Loading the biopolymer with vitamin B12

The films were loaded with vitamin B12 according to the concentrations and volumes given in the Table 2. Vitamin B12 release was determined by using ultraviolet-visible (UV-Vis) spectrometer.

Results and Discussion

UV-Vis Spectrophotometer Analyses

In present study, UV-Vis spectrophotometer was used to examine the vitamin B12 release from biopolymer. By scanning the wavelength in the range of 300-600 nm, the spectrum (Figure 1) was obtained and the maximum absorption wavelength was determined as 360 nm. Then vitamin B12 release was measured at this wavelength.

Figure 1. UV-Vis spectrum of vitamin B12.

Fourier-transform infrared (FT-IR) spectroscopy analysis

Fourier-transform infrared (FT-IR) spectroscopy was used for determining the functional groups that helped define the surface chemistry, chemical properties of the synthesized biopolymer. The FT-IR spectrum of chitosan, chitosan–grafted PAA, and vitamin B12- loaded chitosan–grafted PAA were shown in Figure 2.

Figure 2. FT-IR spectrum of chitosan, chitosan–grafted PAA, and vitamin B12- loaded chitosan–grafted PAA.

Chitosan peaks at 3,287 cm-1 were assigned to O-H stretching and at 2,864 cm-1 to CH stretching. Bands at 1,647 cm-1, 1,590 cm-1 and 1,322 cm-1 indicated amide I, amide II, and amide III, respectively. Bridge O stretching, C-O stretching, and pyranoid ring stretching were revealed at 1,151 cm−1, 1,061 cm−1, and 890 cm−1. Band at 1,026 cm-1 was derived from C-O stretching of the primary -OH. Asymmetric stretching of C–O–C was seen at 1,154 cm-1. After the reaction, these characteristic absorption bands were either weakened or masked by other absorption bands, which meant that these were involved in the co-polymerization reaction. In the spectrum of chitosan-grafted PAA, some new absorption peaks appeared in addition to the characteristic peaks of chitosan. Peaks at 1,658 cm-1, 1,551 cm-1, and 1,435 cm-1 were characteristic peaks of PAA and were stretchings of C-H vibration, asymmetric -COO stretching vibration, and C-H bending vibration, respectively. The peak at 1,658 cm-1 matched carboxyl absorption by grafted PAA, and the peaks at 809 cm-1 and 620 cm-1 were also characteristics of PAA. Also, the bands at 1,551 cm-1 and 1,409 cm-1 matched the sodium carboxyl group. This demonstrated that the acrylic acid in the polymer was grafted onto chitosan and could be assigned to asymmetric and symmetric stretching vibrations of the COO- anion groups. This indicated that the carboxylic groups of PAA dissociated into COO- groups, which formed complexes with protonated chitosan amino groups via electrostatic interaction to form polyelectrolyte complex during the polymerization procedure. The main bands of vitamin B12 were in the range of 2,800-3,500 cm-1 and 1,000 cm-1-1,750 cm-1. Band widening and intensity reductions in the FT-IR spectra between the unloaded film and the B12- loaded film established chemical bond formation between the vitamin B12 molecules and the biopolymer film at 2,940 cm-1 and 2,920 cm-1 peaks (Zhang et al., 2006). The peak of the -CONH2 group was observed at 1,655 cm-1 (Qui et al., 2004). Peaks at 1,555 cm-1 represented the peaks of the amide N-H bands (Dengre et al., 2000). There were peaks of CH2 swing bands at 1,410 cm-1 (Tangpasuthadol et al., 2003). Peaks of N-C tensile vibration were observed at 1,295 cm-1 (Osman and Aro, 2003). Peak of the C-O stretching vibration in C-O-C bands was observed at 1,160 cm-1 (Yuan et al., 2010). A peak of skeletal stress is observed at 1,075 cm-1 (Santi et al., 2012). The band at 1,025 cm-1 in the FTIR spectrum of chitosan film, corresponding to the wagging vibration of the saccharide structure (Boonsongit et al., 2008).

Thermogravimetric Analysis and Differential Thermal Analysis System (TGA-DTA)

The thermal stability of biopolymers and vitamin B12-loaded biopolymers was examined using TGA from room temperature to 600 °C at a heating rate of N2 at 4 °C min–1. The percentage of mass loss of chitosan-grafted PAA and vitamin B12-loaded chitosan-grafted PAA is shown in Figure 3. Major mass loss at up to 160 °C was related to the evaporation of water molecules. In the second step between the 160 °C and 275 °C, the decay of -COOH groups was observed. In the third step (275 °C490 °C), where mass loss was evident, it was assigned to deconstruction. Polymers are the basic inputs used in the production of materials, such as plastic, rubber, fiber, paint, and adhesives that are used frequently in every aspect of daily life. Polymers that occur spontaneously in nature are called natural polymers, and most of the natural polymers are found in living structures (Sacak, 2005). Examples of natural polymers are polysaccharides, such as lignin, cellulose, hemicellulose, alginate, chitin, chitosan, and heparin, and proteins, such as collagen, fibrin, keratin, and eggshell membrane (Mogosanu and Grumezescu, 2013; Sandak et al., 2014). The TGA technique is widely used to study the fundamental stability and properties of thermal decomposition of polymers. The TGA-differential thermal calorimetry (DSC) studies of other chitosan-based polymers reported a decomposition temperature of 310 °C (Cardenas et al., 1992; Kittur et al., 2002). These results were similar to results of the present study.

Figure 3. TGA thermograms of chitosan-grafted PAA and vitamin B12-loaded chitosan-grafted PAA.

The DTA results of chitosan-grafted PAA and vitamin B12-loaded chitosan-grafted PAA are shown in Figure 4. In Figure 4, it was observed in Figure 4 that the decomposition was proportionally higher at endothermic peak at 75°C and at TGA thermogram (Figure 3). This supported that vitamin B12 was loaded on chitosan-grafted PAA.

Figure 4. DTA thermograms of chitosan-grafted PAA and vitamin B12-loaded chitosan-grafted PAA.

Scanning Electron Microscope (SEM)-Energy Dispersive Spectroscopy (EDS) Analyses

The SEM-EDS images of chitosan-grafted PAA and vitamin B12-loaded chitosan-grafted PAA are shown in Figures 5 and 6, respectively. From the EDS images shown in Figure 6, it was interpreted that vitamin B12 was bound on the biopolymer. While the peaks of Co element were not dicovered in the EDS image of chitosan-grafted PAA (Figure 5), the same were checked after vitamin B12 was loaded on chitosan-grafted PAA (Figure 6).

Figure 5. EDS image of chitosan-grafted PAA.

Figure 6. EDS image of vitamin B12-loaded, chitosan-grafted PAA.

Scanning Electron Misroscopy in all studies and applications involving polymers has been used to examine cracks or scratches on the surfaces, phase boundaries in the materials, agglomeration of the support and additive materials, the attraction between the materials and the surface roughness of materials. Roughnesses, such as indentations and protrusions on the surface, is very important as it affects the wetting feature intensely. This property, which is known as the wettability, which features in solids and is similar to the tendency of fluids to stick to a surface, is directly related to the structure of the surface.

Ideal Model Choice for Vitamin B12 Release

In order to optimize the range and levels of variables that affect the vitamin B12 release process from biopolymers and to elucidate the nature of the response surface in the experimental design, the CCD model coupled with RSM was selected and developed considering all the important interactions. In the study, it was understood that the CCD model was quadratic and that there was no need for any power transformation. Among the various applications, the use of biopolymers as drug carriers is one of the most promising applications. High loading capacity, encapsulation efficiency, simultaneous application of various treatments, ease of operation, and low cost are the features that make the use of nanofibers produced by electrospinning method attractive in this field (Hu et al., 2014). Controlled drug release is a method in which the active substance is designed to be delivered in the desired amount at a certain speed in the system for the desired time (Gur and Taskin, 2004). In classical drug release, the active substance is released suddenly, and the toxic drug concentration in the plasma rises above its value: however, this may cause undesirable adverse effects to the patient. In drug release with nanofibers, the drug substance can be added to the electrospinning solution or it can be produced by encapsulation. Thus, nanofibers are used as both a drug carrier and as a drug delivery system (Celik, 2013). Among the various applications, the use of biopolymers as drug carriers is one of the most promising applications. High loading capacity, encapsulation efficiency, simultaneous application of various treatments, ease of operation, and low cost are the features that make the use of nanofibers produced by electrospinning method attractive to this field (Hu et al., 2014). In drug release with nanofibers, the drug substance is added to the electrospinning solution or it is produced by encapsulation. The literature demostrated the release of vitamin B12 are examined, it can be seen that the release of vitamin B12 from nanofiber systems developed with different polymers. Today, many studies Show the controlled release of vitamin B12 (Baskakova et al., 2016; Mendes et al., 2016).

The ANOVA results and regression coefficients are presented in Table 1 and The results showed that the contribution of a quadratic model was significant (p<0.0001) fort he release of vitamin B12. Fisher’s test” shows that larger F-values and smaller p-values were more significant than the suggested model terms. In the present investigation, p < 0.05 was considered as statistically significant at the 95% confidence level (95% Cl), and F=35.08 indicated that the model was significant. If probe >F <0.05, it showed that the model term was statistically insignificant for the model and probe>F>0.05 showed that term was statistically insignificant fort he selected model. Based on the preferred quadratic, some model terms, such as X1, X2, X3, and X4, were statistically significant for vitamin B12 release from biopolymer. Also, the variation of the data around the model that matched the experimental data was expressed as the model’s lack of fit (LOF). The p values of model’s LOF measured the fit of the model and indicated that the LOF was not significant with respect to pure error. This value was a sufficient test for the adequacy of model fit without the effects of additional higher-order terms. The LOF value obtained using ANOVA was 0.0670. These LOF p values of the vitamin B12 release method from biopolymer confirmed the applicability of good reaction settlement. It also showed that the number of experiments performed was sufficient to determine the effects of independent variables on vitamin B12 loading for drug release from biopolymer. Since the R2 and adjusted R2 values describe the percentage of variation in response, the validity of the polynomial models was evaluated with these values. As the R2 value of the quadratic model used was obtained as 0.9704, it was concluded that 97.04% of the values estimated by the model matched the experimental values of the release behavior of vitamin B12-loaded biopolymers (Figure 7).

Figure 7. The overlap ratio of the estimated and actual values obtained for vitamin B12-loaded biopolymers.

The sensitivity term (AP) measures the signal-to-noise ratio (S/N) and it is desirable that this ratio must be greater than 4. This value was obtained as 22.004, which is a clearly indicated that the model was providing sufficient signal. In the light of preliminary experimental studies, four critical parameters affecting release of vitamin B12 from biopolymers were selected as independent variables, and drug release was considered as an dependent variable. As observed in Table 1, the quadratic model with the highest R2 (0.9704) value was preferred according to the statistical test results of the model used. In order to express the relationship between the independent variables and responses, the experimental data were expressed with a quadratic polynomial mathematical equation. The quadratic mathematical equation (Equation 1) of the model in question was derived the coded factors.

Release (%) = –122.44 + 13.58X1 + 1.14X2 + 0.95X3 +84.91X4 + 0.037X1X2 + 0.01X1X3 – 0.79X1X4 – 2.47917E – 003X2X3 – 0.14 X2X4 – 0.11X3X4 – 1.47X12 – 4.95X22 – 2.28X32 – 13.36X42.

The best method to determine the optimum point where the parameters selected as independent variables had the maximum effect and to obtain the highest performance were the arrangement of two-dimensional (2D) and three-dimensional (3D) surface graphs of vitamin B12 release from biopolymer. The effects and interactions of each parameter on the vitamin B12 release biopolymers are presented in Figure 8. The 3D response surface plots derived from CCD were used to define the response maximum, middle, and minimum points of the response.

Figure 8. Interaction between pH and initial concentration. (A) 3D graph and (B) 2D graph.

Figures 8A and 8B represent 3D and 2D response surface plots of the effect of initial concentration and pH on vitamin B12 release efficiency from biopolymers. When pH increased from 3 to 7, release of vitamin B12 from the biopolymers increased up to pH 5, followed by a partial decrease in vitamin B12 release as pH increased to 7. While the initial concentration had a significant effect on vitamin B12 release from biopolymers, it was observed that the effect of the interaction between the initial concentration and pH was not statistically significant (p>0.005). In a study conducted by Nath et al. (2020), the authors investigated an acrylic acid-grafted gelatin/ layered double hydroxide (LDH)- based biocompatible hydrogel with pH-controlled vitamin B12 release. The study reported that a certain increase in the release of vitamin B12 from the biopolymers with increased pH values, in parallel with the results of our study, and a partial decrease in the release of vitamin B12 from the biopolymers when the pH value increased to 7.

In a study conducted by Bajpai and Dubey (2005), it was determined that vitamin B12 released in the environment was 8.6% ± 2.1% and 83.2% ± 4.8% at pH 1.2 and 6.8, respectively.

The 3D and 2D response surface plots of the effect of release time and pH of vitamin B12 release efficiency are shown in Figures 9A and 9B. While the increase in the release time caused a significant increase in the release of vitamin B12 from the biopolymers, its release from the biopolymers increased with increase in pH increased from 3 to 5, but a partial decrease was observed after pH 5. It was observed that the effect of the interaction between pH and release time was not statistically significant (p > 0.05).

Figure 9. Interaction between pH and release time: (A) 3D graph and (B) 2D graph.

The effect of solution volume and pH on release of vitamin B12 is presented in Figures 10A,B. While increase in the solution volume caused a significant increase in the release of vitamin B12 from the biopolymers when pH increased from 3 to 5, the release decreased partially with increase in pH above 5. However, the effect of the interrraction between pH and solution volume was not statistically significant (p > 0.05). A study conducted by Moradi et al., (2020) reported that the initial volume affected the release of vitamin B12. The study determined the optimal initial concentration of vitamin B12 as 1,000 mol m–3.

Figure 10. Effect of interaction between pH and solution volüme on vitamin B12 release efficiency: (A) 3D graph and (B) 2D graph.

The 2D and 3D response surface plots of the effect of release time and initial concentration of release of vitamin B12 are presented in Figures 11A,B. It was determined that increase in release time and initial concentration positively affected the release of vitamin B12. However, using the ANOVA, it was determined that the interactive effect of both variables was not statistically significant (p>0.05). A study determined that the drug concentration loaded on the carrier increased more than the desired amount for a certain time in order to control the optimum drug release from the carrier to the body, and at the same time, the speed of the boundary layer was proportional to the rate of water infusion into the hydrogel. Optimal simulation was obtained with an initial vitamin B12 concentration of 1,000 mol m–3, hydrogel membrane thickness of 0.0004 mm, and a constant velocity of 2 x 10–11 m s–1 (Moradi et al., 2020). In the present study, the time required to achieve equilibrium concentration for a stationary boundary of the system was 1,20, 000 s, while it was 2,10,000 s for a moving boundary with a constant velocity of 2×10–11 m s-1.

Figure 11. Effect of interaction between release time and initial concentration on vitamin B12 release efficiency: (A) 3D graph and (B) 2D graph.

The effect of solution volume and initial concentration on vitamin B12 release efficiency are presented in Figures 12A, B. Both variables contributed positively to vitamin B12 release by increasing the solution volume and initial concentration. However, ANOVA results determined that the interactive effect of both variables was not statistically significant (p > 0.05). The study conducted by Moradi et al., (2020) reported that the initial concentration was effective on vitamin B12 release, and the optimal initial concentration for vitamin B12 was determined as 1,000 mol m-3.

Figure 12. Effect of interaction between solution volume and initial concentration on vitamin B12 release efficiency: (A) 3D graph and (B) 2D graph.

The 3D and 2D response surface plots of the effect of solution volume and release time on vitamin B12 release efficiency are presented in Figures 13A, B. Increase in solution volume and release time contributed positively to vitamin B12 release from biopolymers. Also, ANOVA results demostrated that the interactive effect of solution volume and release time was statistically significant (p < 0.01). The study conducted by de Abreu Figueiredo et al. (2020), it was reported that solution volume and release time were associated with release of drug from biopolymers. Nath et al. (2020) examined the effect of acrylic acid-grafted gelatin/LDH-based biocompatible hydrogel with the pH-controlled vitamin B12 release in their study. The researchers reported that the volume of the solution and the release time were related to the release of vitamin B12 from the biopolymers.

Figure 13. Effect of interaction between solution volume and release time on vitamin B12 release efficiency: (A) 3D graph and (B) 2D graph.

In Figure 14A, a Box-Cox graph for vitamin B12 release was obtained to control the value of lambda (λ). The Box-Cox plot is a preferred diagnostic plot drawn for estimating any necessary transformation of the experimental value to increase the significance of the model. As observed from the power conversion graph, it was confirmed by the model that no conversion was needed according to the obtained λ value (λ = 1; the most ideal value of λ is 0.78).

Figure 14. Diagnostic plots: power conversion and disorder plot for vitamin B12 release from biopolymers at optimum conditions.

A perturbation graph (Figure 14B) was drawn to compare the influence of all factors on the optimum conditions for the release of vitamin B12 release from biopolymers. The graph was also used to analyze the variations in factors as well as the combined effect of all factors on a process. In addition, a power conversion plot for vitamin B12 release was obtained to control λ value. The power conversion plot is often a preferred diagnostic plot drawn to estimate any necessary conversion of the experimental value to increase the significance of the model. As discovered from the power conversion graph, it was model that there was no need for any conversion according to the obtained λ value, that is, λ = 1.

In order to select the optimal model and the maximum desirability function obtained, the most important statistical metrics, such as the lowest LOF and the highest p-value R2, and F-value, were searched. The desirability value of the optimization process was found as 1.0, and is presented in Figure 15. The lower and upper limits of vitamin B12 release of the quadratic model, which were obtained at the end of the experimental study, are presented in Table 1, and the significance level determined was 3.

Figure 15. Desirability plot for vitamin B12 release from biopolymers at optimum conditions.

Based on the possible global solutions (Table 3), validation experiments were performed to support the optimized data from numerical modelling under optimum conditions.

Table 3. Possible solutions.

Number pH Initial concentration Release time(min) Solution volume(%) Release % Std.Error
(Release [%])
1. 4.241 54.039 117.066 2.084 101.258 1.627 1.000 Selected
2. 6.560 74.436 111.860 1.938 101.890 1.597 1.000
3. 5.273 72.369 83.579 2.138 100.370 1.429 1.000
4. 6.033 54.903 108.754 2.240 100.587 1.579 1.000
5. 4.604 72.534 106.784 2.058 102.901 1.436 1.000
6. 5.536 68.508 113.608 1.892 103.286 1.437 1.000
7. 5.300 67.250 94.500 1.863 100.094 1.468 1.000
8. 6.662 77.469 117.750 2.373 100.753 2.172 1.000
9. 4.079 51.111 119.889 2.489 100.050 2.227 1.000
10. 5.579 71.394 94.881 2.355 101.804 1.452 1.000
11. 4.418 57.967 104.873 2.403 101.113 1.518 1.000
12. 6.752 79.317 116.573 1.899 101.805 1.959 1.000
13. 5.424 51.930 115.446 2.473 101.011 1.935 1.000
14. 5.153 69.467 90.213 2.148 101.335 1.436 1.000
15. 4.446 78.783 111.762 2.429 101.497 1.852 1.000
16. 5.632 74.078 94.113 1.820 100.441 1.430 1.000
17. 4.854 75.914 111.612 2.452 102.348 1.712 1.000
18. 4.429 71.344 117.734 1.871 102.417 1.483 1.000
19. 4.287 64.830 107.867 2.303 101.983 1.464 1.000
20. 5.709 68.688 93.893 2.289 101.478 1.433 1.000
21. 4.090 61.704 106.371 2.386 100.861 1.510 1.000
22. 6.059 68.664 107.579 1.894 101.891 1.433 1.000
23. 4.815 71.770 111.034 2.481 102.220 1.615 1.000
24. 5.171 79.770 91.874 2.372 101.631 1.620 1.000
25. 5.581 73.361 108.515 2.450 102.446 1.613 1.000

The values obtained from the experimental design were transferred into the CCD system, the ramp chart of the optimum points of the independent variables in vitamin B12 release from biopolymers is given in Figure 16. By presenting the ranges of all variables in the figure, the desirability value obtained was as high as 1.0.

Figure 16. Ramp chart of statistically optimized factors for vitamin B12 release from biopolymers.

When the approval report of the quadratic model used was examined at the 95% Cl, the standard deviation obtained was 0.000, and the model was significant and valid.


In recent years, there has been an increase in studies on the development of systems that can direct the drug to the target areas of the body and control the long-term drug release rate have increased. These developed systems have been widely used in various fields, such as medicine, physic, biology, biomedicine, and gene engineering. Controlled release systems are the systems that allow the release of active substances to be released to the target areas at a predetermined rate and at specific time intervals. The main purpose of these systems is to maintain the contant concentration of active substances. Polymers are one of the most used materials as a controlled drug system. The release of the active substances is closely related to the properties of the polymer used as well as physiological conditions, such as pH, ionic strength, temperature, and enzyme. In classical applications, possibility exists that the drug concentration in the blood increase above the safe level. Quantity below the mentioned effective level and above the safe level represents the amount of wasted active substance. The main advantages of controlled drug release system over the conventional drug applications are as follows: its abilit to maintain a contant drug level constant at a therapeutic rate; elimination of possible adverse effects in the body because the drug directly affects the desired area; and economical aspect of the drugs used, that is, no need to the use of drugs more than the amount required for treatment.

In addition to these advantages of controlled drug release systems, the following points are worthy of attention: (1) the polymer carrying the drug or the degradation products of this polymer should not have toxicities, that is it should release the drug speedily and must not pose a danger to the body; (2) there must not be any discomfort from the system or the way it is applied to the body; (3) the polymeric material forming the system itself and its preparation should not be expensive; (4) no cracks in the structure of the system that could cause sudden release of the drug; and (5) used polymer’s mechanical properties should be good.

In terms of drug delivery, drug delivery systems can be broadly divided into three groups: (1) delayed release system: this system releases drug at the targeted area of the body because of the elimination of the adverse effects of the drug; (2) sustained release system: it ensures that the drug is released continuously and maintains a constant level in the blood plasma; and (3) controlled release system: in this system, drug is released in response to a variable peresent in the external environment (Baker, 2012).

In membrane systems, the polymers used are in the form of sheets, films, capsules, or microcapsules. The drug to be released is placed in these membranes in either dissolved or dispersed form (Blagoeva and Assen, 2006). During the release, the drug diffuses through the membrane to rise to the outer surface of the membrane. Diffusion of the drug across the membrane is the rate-determining step.

Matrix systems are prepared by dissolving the drug in a solid polymer and dispersing it homogeneously. It is easy and inexpensive to produce; however, since there is a first-degree release, the rate of release and thus the amount of drug given to the outside environment decrease constantly. This is a disadvantage of the system (Reddy et al., 2017).

Worn systems in the body indicate hydrolytically or enzymatically degradable bonds within the structure. For this reason, these systems are also called “biodegradable systems”. Because of the biodegradation of the said bonds, the polymer is eroded and the drug is released. The biggest advantage of these systems is that these can be eliminated from the body in natural manner without the need for surgical intervention. However, here, the material and its decomposition products should not have any health problems (Lee and Yeo, 2015).

In chain-mounted systems, the drug molecules planned to be sent to the body are bound to the polymer chain by covalent bonds that are broken down by enzymatic or hydrolytic process and the drug is released in the body. The appropriate designing of the polymers breaks down the bonds, thereby controlling the release rate of drug. These systems are generally used for short-term drug release applications. The biggest advantage of the system, compared to other systems, is that it can contain about 80% drug by weight (Kismir, 2011). In swelling-controlled systems, cross-linked biopolymers form the basis of these systems. Drug molecules in biopolymers diffuse into the external environment by swelling of the biopolymers in the release medium (water or biological fluid). Here, the chemical structure of the biopolymer and the cross-link ratio, in other words the pore size, play an important role and determine the rate and amount of drug released (Kismir, 2011). The controlled drug release systems are prepared by taking advantage of the environmental intumescent biopolymer properties, which can release drugs sensitive to pH, temperature, light, and magnetic and electric fields. Here again, drug release takes place via the swelling mechanism (Kismir, 2011). Osmotic controlled systems consist of a semi-permeable membrane and the drug placed in this membrane. When they are placed in water or a biological fluid, the molecules in the environment penetrate through the pores of the semipermeable membrane and dissolve inside the drug molecules inside. However, to diffuse through the membrane, a hole must be drilled from a suitable part of the system. For this, the laser method is used. Because the drug concentration in these systems is very high (above the saturation limit), the release is usually of zero-order and osmotically controlled (Kismir, 2011).

pH-responsive biopolymers contain side groups and ionizable groups in the cross-link that accept or donate protons in response to the environmental pH. In this case, changing the pH across the pKa threshold of a polymer causes a rapid change in the net charge and hydrodynamic volume of the polymer chains. In other words, the presence of a high-loaded structure causes the mesh to swell because of high-load repellency and ability of biopolymer to change from collapsed to swollen state. The osmotic pressure created by the presence of mobile counterions also helps to explain the increase in the hydrodynamic volume of the biopolymer network chains. It is particularly interesting to exploit pH differences in the human body (e.g.throughout the GIT) to achieve delivery of a drug to the targeted site using polymers that respond to pH changes. In addition, in anticancer drug delivery, the release of drugs can be triggered by the acidic extracellular pH of tumors; this contributes to higher efficacy and lower toxicity to peripheral tissues. PAA is generally used in drug delivery systems targeting the intestine. Owing to the presence of ionizable -COOH groups, this polyelectrolyte structure is protonated so that it is uncharged at low pH and negatively charged at high pH. The presence of a protonated structure at low pH (as in gastric media) eliminates electrostatic repulsive forces and contributes to the formation of a tighter structure (Calejo, 2013).

Formation of a low elasticity network is also promoted by the formation of hydrogen bonds. When the pH rises above the pKa of the biopolymer, the dominant negative charges cause swelling of the network with high charge repellency and release of the drug. Accordingly, because of higher pH of 7.4 in the intestinal environment, the drug is released from PAA polymer network (Calejo, 2013). For other applications, it can also specifically trigger drug release in response to acidic environments. Polybases with amino groups, such as pDMAEMA and poly(amino esters), are deprotonated at neutral pH but charged by acquiring protons at acidic pH. This is particularly effective in the delivery of anticancer. Since the extracellular environment of solid tumors is weakly acidic (pH < 6.5), the pH in endosomes and lysosomes of cancer cells is lower (pH 4.0–6.0). The particles formed by polybases are stable at physiological pH. However, in tumor tissues or their intracellular compartments, the particles are charged positively and dissolved rapidly in response to lower pH, thereby releasing the cytotoxic drug. In normal tissues, the slightly acidic environment of endosomes is used to trigger cargo release in gene delivery applications. Polyethyleneimine (pEI) and poly(L-lysine) (pLL) are two well-known transporters in nonviral gene delivery system (Calejo, 2013). The gels that shrink in gastric pH (pH<2.0) swell in the intestinal environment (pH>7.0) to release the drug. In another application made in the opposite direction with this process, bad-tasting drugs are released with the help of polymers that swell at a low pH. Because the polymer has a low swelling level in at the neutral pH of the mouth (pH 7.0), drug release due to the environment does not take place. Drug release is assured in the acidic medium of the stomach with low pH (Calejo, 2013; Ilmain et al., 1991). pH-sensitive polymers are smart polymers that can respond to pH change as a variable condition. Acidic or basic ionizable groups are present in the structures of these polymers. These ionizable groups in the structure of the pH-sensitive polymers take or donate protons depending on the charge in pH change. This causes changes in the polymer chain to change, resulting in swelling or shrinkage of the polymers (Reyes-Ortega, 2014).

Acidic side groups of pH-sensitive polymers are ionized in neutral or alkaline solutions. The ionization of acidic side groups depends on the logarithmic value of the acid dissociation constant of a polymer, known as pKa. In cases where the pH value of the medium is greater than the pKa value, ionization of acidic groups takes place. Owing to the electrostatic repulsion forces between the ionized groups, the molecular conformation of the polymer changes, resulting in the swelling of the polymer. These polymers are widely used in drug release (Grainger and El-Sayed, 2010).

The second class of pH-sensitive polymers are the polymers containing basic functional groups, that is, cationic groups. Poly(2-(dimethylamino)ethyl methacrylate) and poly(vinyl amine) are widely used pH-sensitive cationic polymers. Unlike polymers containing acidic groups, these polymers show a structure change at pH 8 or a higher pH value. However, as soon as the pH value drops below the pKb (basic ionization constant) value of the cationic group-containing polymer, the cationic groups absorb protons and the polymer swells because the positively charged groups repel each other with electrostatic interaction (Grainger and El-Sayed, 2010; Simsek, 2016).

In the present study, pH-sensitive biopolymers were developed and their use in colon-specific drug release was investigated. Based on these investigations, by loading of vitamin B12 onto pH-sensitive biopolymers synthesized with chitosan, and its release throughout the intestinal tract was studied. The synthesized biopolymers were characterized using FT-IR, TGA-DTA, and scanning electron microscopy-energy-dispersive x-ray spectrometry (SEM_EDS) techniques. Biopolymers, whose characterization was completed and produced in a film form, were used for drug release by loading vitamin B12. The following results were obtained by performing the experimental study design and multi-parameter optimization.

The proposed mathematical models (Equation 1) also provided critical analyses for the interactive effects of selected independent variables on the vitamin B12 release process from pH-sensitive biopolymers. It was concluded that the values of R2 values (for chitosan-synthesized biopolymer material: R2=0.9704, and R2adjusted=0.9427) obtained using (ANOVA) were of satisfactory level and the overlap ratios of the estimated and actual values were more than 97% of the values predicted by the model match the vitamin B12 release values from biopolymers. Based on the 2D and 3D plots, it was observed that the percentage release of vitamin B12 from pH-sensitive biopolymers increased significantly with increase in pH from 3 to along with increase in initial concentration.

The release conditions of vitamin B12 from the biofilm were determined at pH 4.2, with initial concentration of vitamin B12 being 54.0 mg L-1, the release time being 117 min, and the solution volume as 2.1 mL. ANOVA results showed that the determination coefficients for the use of synthesized biofilm in vitamin B12 release were high and the R2 = 0.9704.

Data from the proposed three-factor CCD combined with RSM confirmed that optimization was an effective approach to model vitamin B12 release from pH-sensitive biopolymer, understanding the relationships between independent and response variables and maximizing process efficiency.

Hence, biopolymer materials synthesized, characterized, and used are good candidates for the release of drugs because of their releasing capability.

Author Contributions

Conceptualization, Onal A. and Demir H..; methodology, Onal A., Ince O.K, Ince M., Demir, H.; software, Onal, A.,Ince, M.; validation, Onal,A.,Ince, M.;Demir H.; formal analysis, Onal, A.Ince, O.K., Demir, H.; investigation, Onal, A.; resources, Onal, A.;Ince, O.K., Ince M., data curation, Onal, A., Ince O.K.; writing—original draft preparation, Demir, H.; Ince M.; writing—review and editing, Demir, H., Ince M., Ince, O.K.; supervision, Demir H., Ince, O.K


This research received no external funding.

Conflicts of Interest

The authors declare no conflict of interest.


Ayhan F., Gülsu, A., and Ayhan H. 2007. Quartz crystal piezo electrode surface modification for gold nano-particles immobilization. Hacettepe J. Biol and Chem. 35(3): 203–208.

Bajpai S. K., and Sharma, S. 2004. Investigation of swelling/degradation behaviour of alginate beads crosslinked with Ca2+ and Ba2+ ions. React and Funct Polym. 59(2): 129–140. 10.1016/j.reactfunctpolym.2004.01.002

Bajpai S.K., and Dubey S. 2005. In vitro dissolution studies for release of vitamin B12 from poly(N-vinyl-2-pyrrolidone-co-acrylic acid) hydrogels. React and Funct Polym. 62(1): 93–104. 10.1016/j.reactfunctpolym.2004.09.004

Baker S.B. 2012. Kontrollu ilac salım hidrojel sistemleri ve uygulama calısmaları, Master’s thesis, Osmangazi University, Eskisehir

Baskakova A., Awwad S., Jiménez J. Q., Gill H., Novikov O., Khaw P.T., et al. 2016. Electrospun formulations of acyclovir, ciprofloxacin and cyanocobalamin for ocular drug delivery. Int J Pharm. 502(1–2): 208–218. 10.1016/j.ijpharm.2016.02.015

Blagoeva R., and Assen, N. 2006. Monolithic controlled delivery systems: Part I. Basic characteristics and mechanisms. Int J Bioautom. 4: 80–88.

Boonsongrit Y., Mueller W.B., and Mitrevej A. 2008. Characterization of drugchitosan interaction by H NMR, FTIR and isothermal titration calorimetry. Eur J Pharm Biopharm. 69: 388–395. 10.1016/j.ejpb.2007.11.008

Calejo M. T. R. 2013. Thermoresponsive Polymers in Controlled Drug Delivery and Gene Delivery. Doctoral thesis, School of Pharmacy, Faculty of Mathematics and Natural Sciences, University of Oslo, Oslo, Norway, 140 p.

Cardenas G., Bernal L., and Tagle L.H. 1992. Thermogravimetric studies of chitosan derivatives. Acta. 195: 33–38. 10.1016/0040-6031(92)80045-X

Çelik G. 2013. Controlled Drug Delivery from the Drug Loaded Nanofiber Systems. Master’s Thesis, Suleyman Demirel Üniversitesi Fen Bilimleri Enstitüsü, Turkey, 113 p.

de Abreu Figueiredo J., de Paula Silva C.R., Oliveira M.P.S., Norcino L. B., Campelo P.H., Botrel D. A., et al. 2020. Microencapsulation by spray chilling in the food industry: Opportunities, challenges and innovatives. Food Res Int. 120: 274–287. 10.1016/j.tifs.2021.12.026

Dengre R., Bajpai M., and Bajpai, S.K. 2000. Release of vitamin B12 from poly (N-vinyl-2-pyrrolidone)-crosslinked polyacrylamide hydrogels: a kinetic study’, J Appl Polym Sci. 76: 1706–1714. 10.1002/(SICI)1097-4628(20000613)76:11<1706::AID-APP12>3.0.CO;2-W

Ghorbanloo M., and Heidary A. 2017. pH sensitive hydrogel based acrylic acid for controlled drug release. Phys Chem Res. 5(1): 185–193. 10.22036/pcr.2017.40420

Grainger S. J., and El-Sayed M.E.H. 2010. Stimuli-Sensitive Particles for Drug Delivery. In: Esmaiel Jabbari and Ali Khademhosseini (Eds.) Biologically- Responsive Hybrid Biomaterials. World Scientific, Singapore, pp. 171–190. 10.22036/pcr.2017.40420

Gupta S., Sharma A., Verma R. S. 2020. Polymers in biosensor devices for cardiovascular applications. Curr Opin Biomed Eng. 13: 69–75. 10.1016/j.cobme.2019.10.002

Gur A.K., and Taskin M. 2004. Metallic biomaterials and biocompability. Firat University Doğu Araştırmaları Dergisi, 2(2): 106–113.

Hu X., Zhou S.L.G., Huang Y., Xie Z., Jing X. 2014. Electrospinning of polymeric nanofibers for drug delivery applications. J Control Release, 10(185): 12–21. 10.1016/j.jconrel.2014.04.018

Ilmain F., Tanaka T., and Kokufuta E. 1991. Volume Transition in a Gel Driven by Hydrogen Bonding. Nature. 349: 400–401. 10.1038/349400a0

Ince M., and Ince O.K. 2019. Hydrocarbon Pollution and its Effect on the Environment. E–Books on Demand EoD). p. 98. 10.5772/intechopen.81556

Jin L., Lu P., Youtl H., Chen Q., and Dong Y. 2009. Vitamin B12 diffusion and binding in crosslinked poly(acrylic acid)s and polys(acrylic acid-co-N-vinyl pyrrolidinone). Int J Pharm. 371(1–2): 82–88. 10.1016/j.ijpharm.2008.12.022.

Jin S.G., Yousaf A.M., Kyeong S.K., Kim D.W., Kim D.S., Kim J.K. et al. 2016. Influence of hydrophilic polymers on functional properties and wound healing efficacy of hydrocolloid based wound dressings. Int J Pharm. 501: 160–166. 10.1016/S0144-8617(01)00320-4

Kismir Y. 2011. Bazı polimerik jellerin hazırlanması ve ilac aktif madde salınımında kullanılması. Doctoral thesis, Istanbul University, İstanbul, Turkey.

Kittur F.S., Prashanth K.V.H., Sankar K.U., and Tharanathan R.N. 2002. Characterization of chitin, chitosan and their carboxymethyl derivatives by differential scanning calorimetry. Carbohydr Polym. 49(2): 185–193. 10.1016/S0144-8617(01)00320-4

Lee J. H., and Yeo, Y. 2015. Controlled drug release from pharmaceutical nanocarriers. Chem Eng Sci. 125: 75–84. 10.1016/j.ces.2014.08.046

Maiti M., Roy A., and Roy S. 2014. Effect of pH and amphiphile concentration on the gel-emulsion of sodium salt of 2-dodecylpyridine-5-boronic acid: Entrapment and release of vitamin B12. Colloids Surf: Physicochemical Eng Aspects. 461: 76. 10.1016/j.colsurfa.2014.07.030

Mendes A.C., Gorzelanny C., Natalia H., Schneider S.W., and Chronakis I.S. 2016. Hybrid electrospun chitosan-phospholipids nanofibers for transdermal drug delivery. Int J Pharm. 510(1): 48–56. /10.1016/j.ijpharm.2016.06.016

Mogosanu, D.G., and Grumezescu, A.M. 2013. Natural and synthetic polymers for wounds and burns dressing. Int J Pharm. 463: 127–136. 10.1016/j.ijpharm.2013.12.015

Moradi H., Khadiv-Parsi P., Sabet J.K., and Esmaeili M. 2020. Simulation of release of vitamin 12 on Hydrogel drug delivery systems. Iran Chem Eng J. 18: 28–35.

Nath J., Ahmed A., Saikia P., Chowdhury A., and Dolui S.K. 2020. Acrylic acid grafted gelatin/LDH based biocompatible hydrogel with pH-controllable release of vitamin B12, Appl Clay Sci. 190: 105569. 10.1016/j.clay.2020.105569

Osman Z., and Aro A.K. 2003. FTIR studies of chitosan acetate based polymer electrolytes. Electrochim Acta. 48: 993–999. 10.1016/S0013-4686(02)00812-5

Qi L., Xu Z., Jiang X., Hu C., and Zou X. 2004. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydr Res. 339: 2693–2700. 10.1016/j.carres.2004.09.007

Reddy A. M., Karthikeyan, R., Vejandla, R.S., Divya G., Babu P.S. 2017. Controlled release matrix drug delivery system–a review. Int J of Allied Med Sci Clin Res (IJAMSCR). 5: 384–390.

Reyes-Ortega F. 2014. pH-responsive polymers: properties, synthesis and applications, Maria Rosa Aguilar (Ed.), Smart Polymers And Their Aplication. Woodhead Publishing, Spain, pp. 45–92. 10.1016/j.ab.2011.08.039

Sacak M. 2005. Polimer Kimyası, Gazi Kitabevi, Ankara, Turkey, 431 p.

Sandak A., Sandak J., Babinski L., Pauliny D., Riggio M. 2014. Spectral analysis of changes to pine and oak wood natural polymers after short-term waterlogging. Polym Degrad Stab. 99: 68–79.

Sarti F., Iqbal J., Müller C., Shahnaz G., Rahmat D., and Schnürch A.B. 2012. Poly (acrylic acid)–cysteine for oral vitamin B12 delivery. Anal Biochem. 420(1): 13–19. 10.1016/j.ab.2011.08.039

Tangpasuthadol, W., Pongchaisirikul, N., and Hoven, V. P. 2003. Surface modification of chitosan films. effects of hydrophobicity on protein adsorption. Carbohydr Res. 338: 937–942. 10.1016/S0008-6215(03)00038-7

Yuan, Q., Hein, S., and Misra R.D.K. 2010. New generation of chitosan-encapsulated ZnO quantum dots loaded with drug: Synthesis, characterization and in vitro drug delivery response. Acta Biomater. 6: 2732–2739. 10.1016/j.actbio.2010.01.025

Zhang C., Ding Y., Ping Q., Yu L. 2006. Novel chitosan-derived nanomaterials and their micelle-forming properties. J Agric Food Chem. 54: 8409–8416. 10.1021/jf061541w