pH responsive cross-linked polymeric matrices based on natural polymers: effect of process variables on swelling characterization and drug delivery properties

Introduction

Scientists have shown enormous interest to improve the bioavailability and biocompatibility of different therapeutic agents. Polymer-based controlled drug delivery system (CDDS) is a better alternative approach because it can be used for targeting therapeutic agents like proteins and small peptides. Hydrogels have great importance in CDDS because of its adjustable, time-dependent swelling behavior and biocompatibility.^1-3

Hydrogels are polymeric 3D networks, hydrophilic in nature, which have the ability to absorb water and other fluids.^4-7 Hydrogels absorb water and other biological fluids due to functional groups such as –CONH, -OH, -SO₃H and CONH₂.⁸ Hydrogels have become progressively significant and are indeed very versatile materials with widely spread applications in various areas such as artificial implantation,⁹ swelling CDDS,¹⁰ artificial skin development,¹¹ wound dressing and humidity sensor.¹² The “Smart gels” and “intelligent gels” are terms that can also be used for them¹³ Receiving, transmitting and mainly responding to various stimuli, are main features of smart materials. Various stimuli include pH, temperature, ionic strength and electric currents.¹⁴

From a biological and medical point of view, pH sensitivity has great importance. The pH values change in the physiological medium of human gut likewise from highly acidic medium (stomach, pH 1-3) to almost neutral conditions (intestine, 6.39-7.49).¹⁵

Various types of crosslinkers are used to increase the mechanical strength of the hydrogels.¹⁶ Crosslinking can be done physically or chemically. Physical crosslinking is a reversible process and creates crosslinking by temporary bonding such as crosslinking through hydrogen bonding or van der Waal’s forces.¹⁷ Mechanical strength and physical integrity of hydrogels are significantly increased by crosslinking. Moreover, crosslinking also makes them water-insoluble.¹⁸ Different crosslinkers are available for natural and synthetic polymers. Moreover various methods have been used to crosslink hydrogels. Glutaraldehyde (GA) can be used for crosslinking polymer containing –OH functional group.^{19, 20} The toxicity of the GA can be removed by using it in a minute quantity, and after crosslinking, its toxic effect can be diminished and the un-crosslinked GA can be removed by washing of hydrogels.

Different types of polymers from natural and synthetic sources are available to prepare pH sensitive controlled release drug delivery system. In contrast to synthetic polymers, natural polymers are generally of low price, biodegradable, biocompatible and nontoxic. Due to these advantages, scientists are nowadays more attracted to synthesize natural polymer-based hydrogel.²¹

Gelatin (Ge) is a polypeptide acquired from collagen by the process of hydrolysis which is obtained from skin of the animals, tissues (connective tissues) and bones.²² It is formed by a different arrangements of amino-acids, with glycine in high amount, proline and hydroxy-proline. In the gelling process of Ge, the role of proline and hydroxylproline is noteworthy.²³ Due to its low price, biodegradation, compatibility and natural origin Ge is used in hydrogels as a natural polymer in great contents.²⁴ At high temperature above (40°C), Ge solutions exist in solution state but on cooling of solution below 30°C, the solution re-assembled to form thermoreversible gels.²⁵

Pectin (Pec) is naturally occurring hydrophilic, polysaccharide found in the cell wall of the higher plants. Pec is the main element in the growth (initial growth) and aging process. It is deducted from apple, plume, gooseberry, cherries, grapes and oranges.^{26, 27} Chemical structure of Pec contains a linear chain of poly-α-(1→4) –Dextro-galacturonic acid with varying level of CH₃ esterification of -C=O (carboxyl group).^{28, 29} Pec is classified into two i.e, low methoxy Pec and high methoxy Pec depending on the amount of esterification of methyl groups.

In the case of high methoxy Pec, due to the presence of large no of methyl groups in the Pec structure, the number of carboxyl groups in turn decreased.³⁰ Low methoxy Pec has the ability of hydrogel formation by reacting with bivalent cationic polymers.³¹ Pec is extensively used as a carrier in drug delivery system.

In the present work, Ge/Pec hydrogels of different composition were developed by crosslinking technique. These hydrogels were crosslinked with GA and mannitol was used as the model drug. The effects of pH, hydrogel composition and crosslinking degree on swelling and release of the model drug were studied in phosphate buffer solution of various pH values. Drug release data was used to investigate the best release mechanism by fitting to various mathematical models. Various structural parameters are used to determine the porosity, strength, solvent interaction and sol-gel fraction of hydrogels. Fourier transform infrared (FTIR) spectroscopy confirmed the structure formation of hydrogels while X-ray diffraction (X-RD) was used to confirm both strength and crystallinity of hydrogels. Differential scanning calorimetry (DSC) confirmed the thermal stability of the synthesized hydrogels. The scanning electron microscopy (SEM) analysis was done to determine the morphology such as porous structure of the hydrogel samples.

Materials and Methods

Synthesis of hybrid network of Ge/Pec hydrogels

In this work, a series of crosslinked hydrogel samples of Ge/Pec with various polymeric compositions and crosslinking ratios were prepared (Fig. 1). Table 1 indicates the feed composition of synthesized hydrogels. The method is briefly described below;

Fig. 1. Presumptive Ge/Pec hydrogel structure.

Table 1. Feed Composition ratio of different formulations of Ge/Pec hydrogels
Name	Ge/100g solution	Pec/100 g solution	Ge/Pec (Wt %)	GA (%)/100 g	GA(g)/100 g solution
S1	12.8	0.8	94.11/5.88	4.0	2.06
S2	14.4	0.8	94.74/5.26	4.0	2.30
S3	16.0	0.8	95.23/4.76	4.0	2.54
S4	12.6	0.9	93.3/6.67	4.0	2.04
S5	12.6	1.06	92.24/7.76	4.0	2.06
S6	12.6	1.20	91.30/8.70	4.0	2.08
S7	12.6	1.20	91.30/8.70	3.8	1.98
S8	12.6	1.20	91.30/8.70	4.2	2.18
S9	12.6	1.20	91.30/8.70	4.6	2.40

Calculated amount of Ge was dissolved in predetermined amounts of 3% v/v glacial acetic acid in distilled water under continuous stirring at 150–200 rpm at a temperature of 60±0.5°C. The desired amount of Pec was weighed and dissolved in a small quantity of 3% v/v glacial acetic acid in distilled water under continuous stirring at 150-200 rpm at temperature 50±0.5°C. Pec solution was added slowly to the already prepared Ge solution and the copolymeric solution was stirred for 25 minutes. GA as a crosslinker was added slowly to Ge and Pec solution under constant stirring at 550 rpm. At this stage, temperature of the solution should be maintained at 37±0.5°C. The volume was made up to 100 g with distilled water. Nitrogen gas bubbling was used to deoxygenate the test tubes for 15 to 20 minutes. After deoxygenating, the glass tubes were strongly fitted using lids. The capped glass tubes were kept for drying at room temperature for 72 hours. After 72 hours the capped tubes were opened. Cylindrical hydrogels were moved out of the glass tubes and were divided into 5 mm discs. The hydrogels discs were placed in Petri dishes at room temperature for drying for minimum 48 hours. The dried discs were placed in distilled water to remove uncrosslinked polymers. The hydrogel discs were washed daily, distilled water was changed continuously until the pH of the old distilled water was kept as same as that of fresh distilled water. After washing, the hydrogel discs were first dried at room temperature and then in an oven at 40–45°C till constant weight.³²

Scanning electron microscopy

The Ge/Pec hydrogels morphology was measured using a scanning electron microscope (Hitachi, S 3000 H, Japan). Samples were attached onto an aluminum mount and sputtered with gold palladium. The coated samples were then observed under SEM at accelerating voltage of 20 and a tilt angle of 30^◦ to observe the microstructure of the unloaded and drug-loaded samples.³⁵

Results

Characterization of Ge/Pec hydrogels

FTIR spectroscopy

In order to assess and check the formation of cross-linked hydrogel networks with GA, samples were analyzed by Fourier transform infrared spectroscopy (FTIR). Fig. 2 indicates the FTIR spectra of (a) Ge (b) Pec (c) mannitol (d) unloaded hydrogel (e) drug-loaded hydrogel. In the FTIR spectra of Ge shown in Fig. 2 (a) the peaks at 1633 cm^-1 and 1440 cm^-1 represents the asymmetric and symmetric stretching vibration of –COO groups respectively. The peaks around 1178 cm^-1 and 1359 cm^-1 corresponds to C=O and C–N and amide II (-NH bending vibration peak). The occurrence of a broad peak at 3479 cm^-1 and 2893 cm^-1 were assigned to –OH and –CH stretching peak. Fig. 2 (b) represents the FTIR spectra of pure Pec. The FTIR spectra of Pec show characteristic peaks at 3972 cm^-1 and 2977 cm^-1 suggested due to the presence of -OH and –CH stretching vibration peaks. The peaks observed at 1689 cm^-1 and 1762 cm^-1 can be attributed to -C=O and carboxymethyl group (CH₂-CO₂H) of Pec. The peaks at 1494 cm^-1 and 1371 cm^-1 were consigned to CH₂ and –OH bending vibration peak. Fig. 2 (c) shows the FTIR spectra of mannitol. The FTIR spectra of the hydrogels shown in Fig. 2 (d) described that there is a reduction in intensity of –OH stretching vibration peak (n = 3250 and 3495) as compared to -OH stretching peaks in pure Pec and Ge. This is attributed to a strong intermolecular interaction between Ge and Pec. The FTIR spectra of drug-loaded hydrogel shown in Fig. 2 (e) indicates that there is no prominent change in the major peaks, which indicates that there is no chemical interaction between polymers and drug-loaded in the hydrogel.

Fig. 2. FTIR spectra of (a) Gelatin (b) Pectin (c) Mannitol (d) unloaded hydrogel (e) drug loaded hydrogel.

X-ray diffraction (XRD) analysis

The XRD pattern of Ge/Pec hydrogel and drug-loaded Ge/Pec hydrogel has been depicted in Fig. 3. The diffractogram of unloaded Ge/Pec hydrogel indicated the peaks at ~16.960^o, 20.140^o, 44.320^o, 64.700^o, 69.120^o, 77.860^o, 78.080^o, (2θ). The diffractogram of drug-loaded Ge/Pec hydrogel indicated peaks at ~ 16.800^o, 20.280^o, 44.460^o, 64.820^o, 69.220^o, 77.960^o, 78.200^o, (2θ), which are nearly same as that of Ge/Pec hydrogel sample without the drug. It means that there is no apparent interaction reported between drug and hydrogel.

Fig. 3. X-ray diffraction (XRD) Analysis of (A) drug loaded Ge/Pec hydrogel sample (B) unloaded Ge/Pec hydrogel samples.

Differential scanning calorimetry

DSC thermograms of unloaded, and drug-loaded hydrogels are presented in Fig. 4. The DSC curve of unloaded Ge/Pec hydrogel shows an endothermic peak which is corresponding to the glass transition temperature of hydrogel between 46°C and 56°C as shown in Fig. 4 (a) . This change in glass transition temperature (Tg) is suggested because of the decomposition of glucosamine units and elimination of bound water in the network chains. Similarly, the DSC spectra of the drug-loaded sample show an initial endothermic peak corresponding to the glass transition temperature (Tg) starting at 46.64°C till 64.24°C as shown in Fig. 4 (b) . This change is also assigned to the water elimination and decomposition of the gel network. However, an increase in Tg of the drug-loaded sample was observed which is suggested to be due to the incorporation of the drug in the interconnected rigid polymer network of hydrogel because of chain entanglements. The drug-loaded hydrogel showed an absence of drug melting peak which indicates molecular dispersion of the drug in the prepared hydrogels.

Fig. 4. DSC thermogram of (a) unloaded hydrogel (b) drug loaded hydrogel.

Scanning electron microscopy

SEM was performed to study the surface and cross-sectional morphology of blank and drug-loaded hydrogel samples respectively. Fig. 5 (A, B and C) indicates the surface and cross-sectional morphology. It is observed that SEM micrographs of the hydrogel samples contain pores or channels. These pores are believed to facilitate the diffusion of solvent and drug particles into the interpenetrating network of the hydrogel.

Fig. 5. Scanning Electron Microscopy (SEM) of hydrogel samples. (A) Surface morphology of blank hydrogel sample (B,C) Cross-sectional morphology of drug loaded hydrogel sample.

The pH-dependent swelling and drug release of Ge/Pec hydrogels

The swelling behavior of the hydrogel samples can be determined by knowing the pH of the solution, pKa of acidic components and pKb of basic components of the polymers. As Ge/Pec hydrogels contain both amino (NH₂) and carboxylic acid (COOH) groups. In order to determine pH-dependent swelling behavior, these samples were immersed in different phosphate buffer solutions of pH 1.2, 5.5 and 7.5.

The pKb of Ge and Pec are 11.5 and 7.5 respectively. The maximum swelling was seen at pH 1.2 due to the presence of -NH₂ group (basic component). This is because, at pH below pKb of basic components, an ionization (protonation) occurs resulting in the formation of –NH₃⁺ ions that display electrostatic repulsion due to the presence of similar cations. The pKa of Ge and Pec are 5.2 and 3.55 respectively. Both Ge and Pec contains -COOH group (acidic component) that becomes COO-(deprotonated) at pH above pKa, Ionization of COOH group at pH 7.5 causes the repulsion of anions due to electrostatic repulsion and thus swelling of the hydrogel occurs. The dynamic and equilibrium swelling ratios are shown in Table 2.

Table 2. Dynamic and equilibrium swelling study of Ge/Pec hydrogels
Sample No	Ge/Pec		pH 1.2		pH 5.5		pH 6.5		pH 7.5
	(Wt %)	Q	Eq	Q	Eq	Q	Eq	Q	Eq
S1	94.11/5.88	3.312	12.51	2.69	3.82	2.056	3.21	2.52	3.96
S2	94.74/5.26	3.304	13.72	2.706	4.04	4	3.97	2.9	4.12
S3	95.23/4.76	4.093	14.32	3.082	4.43	3.1833	4.44	2.92	4.21
S4	93.3/6.67	3.496	12.35	2.877	3.82	2.53	3.70	3.11	4.01
S5	92.24/7.76	3.593	13.01	2.88	3.99	2.63	3.77	3.26	4.03
S6	91.30/8.70	3.315	14.66	2.839	4.08	2.71	3.94	3.145	4.13
S7	91.30/8.70	3.429	13.05	2.938	3.97	2.702	3.71	2.798	4.05
S8	91.30/8.70	3.352	13.79	2.826	3.83	2.652	3.57	2.75	4.01
S9	91.30/8.70	3.075	11.90	2.601	3.78	2.5	3.43	2.695	3.98
Abbreviations: Q, dynamic swelling; Eq, equilibrium swelling.

For drug release study, some samples were selected which showed maximum swelling. Hydrogels with different concentrations of Ge (S₁, S₂ and S₃) and different concentrations of glutaraldehyde (GA) (S₇–S₉) were selected for loading of drug.

Mannitol was chosen as a model drug. The pH effect on drug release behavior was investigated by submerging the mannitol loaded samples in solutions of different pH (pH 1.2, pH 5.5, and pH 7.5). It is observed that maximum release was seen at pH 1.2 as shown in Table 3. It can be correlated with the swelling behavior of the Ge/ Pec hydrogel samples where the maximum swelling was seen at acidic pH. Mannitol was used because it is freely soluble in the water, having short half-life (100 minutes). As Ge/Pec hydrogel showed maximum swelling first at 1.2 pH and then 7.5 pH, mannitol remains undissociated at both acidic and basic pHs due to its strongest acidic pKa of 12.59.

Table 3. Percent (%) mannitol released in different formulations of Ge/Pec hydrogels
Sample codes	pH 1.2	pH 6.5	pH 7.5
S1	80.02	50.21	60.04
S2	82.15	50.21	60.11
S3	81.15	50.37	60.41
S7	81.55	50.87	60.91
S8	80.95	49.97	60.11
S9	80.42	49.43	59.71

Ge concentration effect on swelling and on drug release of Ge/Pec hydrogels

Three formulations of Ge/Pec hydrogels with different concentration of Ge 12.8 g, 14.4 g and 16.0 g keeping Pec and GA concentration constant (4 wt% of Ge and Pec) were prepared and subjected to swelling studies using different pH solutions. Fig. 6 shows the impact of Ge concentration on the dynamic swelling of hydrogels. The numerical data showing the effect of Ge (S₁ to S₃) on both dynamic and equilibrium swelling have been shown in Table 2. The swelling coefficient of prepared hydrogels was increased with the increase in the concentration of Ge. At the pH lower than the pKb value, the Ge polymer chains keep on deprotonated. As a result, the polymer chains hold NH₃⁺ ions, and the cationic repulsion present in the polymer chains could be held responsible for their high swelling. Same results were found by Saarai et al, who synthesized Ge/NaAlg hydrogels.⁴⁷

Fig. 6. Dynamic swelling coefficient of Ge/Pec hydrogels with variable concentrations of Ge (12.8, 14.4 and 16.0g) using GA as crosslinking agent (4 wt% of Ge and Pec) in solutions of different pH in 0.05M USP phosphate buffer at 37^oC. The pH values include; pH 1.2 (▲), pH 5.5 (●), pH 6.5 (♦) and pH 7.5 (■).

Release studies of mannitol were also conducted for samples S₁, S₂ and S₃, with increasing concentrations of Ge, in solutions of variable pH values (pH 1.2, 5.5 and 7.5). The effect of the concentration of Ge on release of mannitol from loaded hydrogel samples has been shown in Fig. 7. It was observed that with the increase in the concentration of Ge drug release was also increased at lower pH value. This is attributed to be due to the increased protonation of amino groups at lower pH values. Further, Figs. 8, 9 and Fig. 10 indicate the % Mannitol release from Ge/Pec hydrogels as a function of time with increasing Ge concentration in hydrogel network.

Fig. 7. Release of Mannitol from Ge/Pec hydrogels using different concentrations of Ge (16 g, 18 g and 20 g) and GA as crosslinking agent (4% of Ge and Pec) in 0.05M USP phosphate buffer solutions of variable pH values.

Fig. 8. Cumulative % Mannitol release using 12.8 g of Ge in the feed composition of Ge/Pec hydrogels at fixed amount of GA as a cross linking agent after 12 hours.

Fig. 9 . Cumulative % Mannitol release using 14.4 g of Ge in the feed composition of Ge/Pec hydrogels at fixed amount of GA as a cross linking agent after 12 hours.

Fig. 10 . Cumulative % Mannitol release using 16.0 g of Ge in the feed composition of Ge/Pec hydrogels at fixed amount of GA as a cross linking agent after 12 hours.

Pec concentration effect on swelling of Ge/Pec hydrogels

Three Ge/Pec hydrogel formulations with varying concentration of Pec (0.9 g, 1.06 g and 1.20 g) keeping Ge and GA concentration constant (4 wt% of both Ge and Pec) were prepared and subjected to swelling studies in solutions of different pH values. In Table 2 the samples (S4 to S6) show the impact of Pec concentration on dynamic and equilibrium swelling ratio. It was observed that at low pH values, the swelling ratio with increased Pec concentration is not significant as compared to swelling ratio at high pH values. This is because at low pH values carboxyl groups (-COOH) of Pec remains protonated and same ions repulsive force is eliminated as a result swelling ratio remains low. At pH values > 4, -COOH are ionized (deprotonated) and electrostatic repulsion of –COO-(anion-anion repulsion) causes the enhancement of swelling. Fig. 11 demonstrates the effect of Pec amount on swelling ratio in different buffer solutions. Similar increase in the swelling has previoulsy reported by Pourjavadi & Barzegar and Yu et al.^{21, 48}

Fig. 11. Dynamic swelling coefficient of Ge/Pec hydrogels with variable concentrations of Pec (0.9 g, 1.06 g and 1.20 g) using GA as crosslinking agent (4 wt% of Ge and Pec) in 0.05M USP phosphate buffer solutions of various pH at 37^oC. The pH values were set as; pH 1.2 (▲), pH 5.5 (●), pH 6.5 (♦) and pH 7.5 (■).

Effect of crosslinking degree on swelling and on drug release of Ge/Pec hydrogels

The swelling and drug release behavior of Ge/Pec hydrogels were also found to be dependent on the contents of GA. In order to investigate the effect of GA on swelling and release behavior of hydrogels, a series of three Ge/Pec hydrogels with different GA concentrations (3.8, 4.2 and 4.6% of Ge and Pec) were prepared as shown in Table 2 and Table 3. In Fig. 12 it was found that gel swelling decreased with the increase in GA concentration owing to the presence of physical entanglements in hydrogels structure. The effect of increasing crosslinking can be explained by low mesh size of the gel network. High cross-linked polymers tend to be less acidic because amino groups are covered and higher crosslinking ratio decreases the ionization process. It was found that at crosslinker higher concentration, the polymer chain relaxation decreased which is responsible for the less swelling of hydrogels. A similar decrease was in the swelling ratio has been reported by Park et al⁴⁹ who prepared PVA/methylcellulose (MC) blend hydrogel and suggested that by increasing GA concentration, the swelling ratio could decrease significantly.

Fig. 12. Dynamic swelling coefficient of Ge/Pec hydrogels with variable concentrations GA as crosslinking agent (3.8 wt%, 4.2 wt% and 4.6 wt% of Ge and Pec) in 0.05M USP phosphate buffer solutions of different pH at 37^oC. The pH values are pH 1.2 (▲), pH 5.5 (●), pH 6.5 (♦) and pH 7.5 (■).

To observe the impact of different cross-linking agent concentrations, drug release studies was conducted in buffer solutions of pH 1.2, 5.5 and 7.5 which demonstrate that increase in GA contents will result in reduction in drug release due to the presence of compact structure of hydrogels as shown in Table 3. Fig. 13 indicates the crosslinking agent concentration effect on drug release in buffer solutions of variable pH values. Further, Figs. 14, 15 and 16 show the cumulative % Mannitol release from Ge/Pec hydrogels as a function of time with increasing GA concentration.

Fig. 13 . Release of Mannitol from Ge/Pec hydrogels using different concentrations of GA (3.8%, 4.2% and 4.6%) in 0.05M USP phosphate buffer solutions of different pH values.

Fig. 14 . Cumulative % Mannitol release from Ge/Pec hydrogels using 3.8% of GA as a cross linking agent after 12 hours at fixed amount of polymeric contents.

Fig. 15 Cumulative % Mannitol release from Ge/Pec hydrogels using 4.2% of GA as a cross linking agent after 12 hours at fixed amount of polymeric contents.

Fig. 16. Cumulative % Mannitol release from Ge/Pec hydrogels using 4.6% of GA as a cross linking agent after 12 hours at fixed amount of polymeric contents.

Sol-gel analysis

To measure the uncross-linked portion of polymer in the hydrogel, the sol-gel analysis was performed. It was observed that gel-fraction of hydrogels increased along with the increased concentrations of Ge, Pec and GA as shown in Table 4. The sol-fraction of hydrogels was found to decrease with the increasing concentration of Ge, Pec and GA. Fig. 17 (a, b, c) demonstrate the impact of polymer concentrations and GA concentration on the gel-fraction of the hydrogel.

Table 4. Gel fraction and Porosity of different samples of Ge/Pec hydrogels
Sample No	Ge/Pec Ratio	Degree of crosslinking GA (wt%)	Gel fraction (%)	Porosity (%)
S1	94.11/5.88	4.0	83.11	30.14
S2	94.74/5.26	4.0	84.64	39.12
S3	95.23/4.76	4.0	85.43	44.5
S4	93.3/6.67	4.0	78.19	19.89
S5	92.24/7.76	4.0	79.01	22.98
S6	91.30/8.70	4.0	80.577	26.79
S7	91.30/8.70	3.8	79.57	25.11
S8	91.30/8.70	4.2	82.75	21.77
S9	91.30/8.70	4.6	84.37	17.68

Fig. 17. Effect of (A) gelatin concentration (B) Pectin concentration (C) GA concentration on gel fraction of gelatin/pectin hydrogels.

Porosity measurement

From results in Table 4, it is found that porosity increases by increasing the contents of Ge and Pec due to increasing of viscosity of the hydrogel solution. Various solutions efficiently prevent the bubbles escaping from the solution, which leads to increase in porosity due to the interconnected channels formation. Increasing GA concentration results in porosity decreases due to the increase physical entanglement between Ge and Pec as shown in Fig. 18. An increase in the concentration of the crosslinking agent resulted in increased in entanglement between polymer chains, leading to a lower porosity.

Fig. 18. Effect of (a) gelatin concentration (b) pectin concentration (c) GA concentration on porosity of Ge/Pec hydrogels

Diffusion coefficient of polymers (D)

Fick’s first law of diffusion was applied during membrane permeation method or sorption and desorption phenomenon.⁵⁰ To measure the solute diffusion into the hydrogel, the diffusion coefficient was used indirectly. It was found that diffusion coefficient was reduced with the increasing of GA concentration. Table 5 shows the diffusion coefficient of all prepared hydrogel samples.

Table 5. Flory-Huggins network parameters of Ge/Pec hydrogels
Samples No	V 2,s	Χ	M c	M r	q	D (cm²/sec)
S1	0.036467	-0.5125	4713.664	94.60281	49.82584	0.159516
S2	0.036238	-0.51242	5129.1	93.95429	54.59144	0.096693
S3	0.034381	-0.51176	5139.356	93.42916	55.00805	0.080312
S4	0.041956	-0.51444	3526.781	95.37452	36.97823	0.149048
S5	0.039419	-0.51354	3710.133	96.46819	38.45965	0.130594
S6	0.038888	-0.51335	4442.316	97.42065	45.59932	0.129911
S7	0.035458	-0.51214	4570.259	96.78327	47.22158	0.135711
S8	0.04245	-0.51462	3234.571	98.05006	32.98897	0.192186
S9	0.043457	-0.51497	3184.885	99.40741	32.03871	0.221298

Physicochemical characterization of Ge/Pec network structure

Molecular weight between crosslinks (Mc) and solvent interaction parameters (χ)

It was observed that increase in contents of Ge results in increased values of molecular weight between crosslinks (M_c). Due to the presence of Ge amino groups in polymer chain higher swelling of polymer was reported. Crosslinked density (N) has also a connection with the concentration of Ge and average molecular weight between crosslinks as shown in Table 5. To investigate the effect of interaction between polymer and solvent, the solvent interaction parameter (χ) was studied. It was observed that the higher the value of (χ) is, the weaker the interaction between solvent and polymer will be.⁵¹

Drug release mechanism

Drug release pattern analysis was studied in different phosphate buffer solutions of pH 1.2, 5.5 and 7.5. The data obtained were fitted to various mathematical models to evaluate the drug release pattern as given in Table 6 and Table 7. Regression coefficient (r) values were used to define the best fits the release data and mechanism. The most appropriate model was based on the selecting criteria by an ideal fit indicated by the values of regression coefficient (r) near to 1. For most of the samples, the value of regression coefficient (r) obtained for zero order release rate constants were found higher than those of first order. It is therefore suggested that drug release from the samples of varying Ge compositions and degree of crosslinking follow zero order release. In Higuchi model the value of regression coefficient (r) at different Ge composition and at a different degree of crosslinking indicated that the drug release mechanism is diffusion controlled.³¹ In Korsmeyer-Peppas model, the n values for different hydrogel samples were found between 0.45 and 1 showing that they follow non-Fickian diffusion mechanism. Tables 8 and 9 indicates the effects of polymer concentration and degree of cross-linking on release exponent (n) respectively.

Table 6. Effect of different contents of Ge on drug release kinetics of Ge/Pec hydrogels in buffer solutions of variable pH values using GA as crosslinking agent (4 wt% of Ge and Pec)
Samples No	Ge contents	pH	Zero order Kinetic		First order kinetics		Higuchi Model
			K 0 (h-1)	R	K 0 (h-1)	r	K 0 (h--1)	r
S1	12.8	1.2	6.449	0.993	0.128	0.980	0.276	0.984
		5.5	4.065	0.988	0.55	0.964	0.169	0.924
		7.5	4.893	0.997	0.073	0.974	0.206	0.958
S2	14.4	1.2	6.475	0.991	0.131	0.976	0.027	0.979
		5.5	4.065	0.988	0.055	0.960	0.169	0.924
		7.5	4.894	0.997	0.073	0.974	0.206	0.958
S3	16.0	1.2	6.413	0.993	0.133	0.974	0.275	0.983
		5.5	4.065	0.988	0.055	0.960	0.169	0.924
		7.5	4.894	0.997	0.067	0.975	0.206	0.958

Table 7. Effect of degree of crosslinking on drug release kinetics of Ge/Pec hydrogels in buffer solutions of variable pH values
Samples No	GA contents	pH	Zero order Kinetic		First order kinetics		Higuchi model
			K 0 (h-1)	r	K 0 (h-1)	r	K 0 (h-1)	r
S7	3.8%	1.2	6.419	0.994	0.116	0.978	0.273	0.970
		5.5	3.744	0.983	0.048	0.957	0.155	0.912
		7.5	4.635	0.993	0.065	0.971	0.194	0.940
S8	4.2%	1.2	6.635	0.994	0.135	0.972	0.279	0.986
		5.5	4.061	0.987	0.055	0.960	0.169	0.924
		7.5	4.891	0.997	0.073	0.974	0.206	0.959
S9	4.6%	1.2	6.591	0.995	0.119	0.964	0.277	0.967
		5.5	3.740	0.983	0.048	0.957	0.155	0.912
		7.5	4.658	0.994	0.065	0.973	0.195	0.942

Table 8. Effect of variable contents of Ge on drug release mechanism of Ge/Pec hydrogels in buffer solutions of variable pH values using GA as crosslinking agent (4 wt% of Ge and Pec)
Sample Codes	Ge content	pH	Release exponent (n)	r	Order of release
S1	26	1.2	0.826	0.995	Non-fickian
		5.5	0.930	0.990	Non-fickian
		7.5	0.947	0.994	Non-fickian
S2	32	1.2	0.782	0.988	Non-fickian
		5.5	0.944	0.989	Non-fickian
		7.5	0.946	0.994	Non-fickian
S3	38	1.2	0.732	0.988	Non-fickian
		5.5	0.930	0.990	Non-fickian
		7.5	0.943	0.994	Non-fickian

Table 9. Effect of degree of crosslinking on release mechanism of Ge/Pec hydrogels in buffer solutions of variable pH values
Samples No	GA content	pH	Release exponent (n)	R	Order of release
S7	3.8%	1.2	0.971	0.990	Non-fickian
		5.5	0.959	0.980	Non-fickian
		7.5	0.992	0.976	Non-fickian
S8	4.2%	1.2	0.880	0.993	Non-fickian
		5.5	0.916	0.987	Non-fickian
		7.5	0.906	0.992	Non-fickian
S9	4.6%	1.2	0.826	0.995	Non-fickian
		5.5	0.930	0.990	Non-fickian
		7.5	0.947	0.994	Non-fickian

Cell culture experiments

In vitro cytocompatibility of Ge/Pec hydrogels

The in vitro cytocompatibility of Ge/Pec hydrogels was evaluated against Vero cells (African green monkey kidney cell lines) using MTT assay. The cytocompatibility was defined as the viability of cells treated with hydrogel formulations (S3, S6 and S9) compared with controls. Fig. 19 shows the in vitro cytocompatibility sketch of the MTT assay. As shown in Fig. 19, there is no significant difference in the % cell viability between the untreated cells and the hydrogel formulation. The hydrogel samples did not show any toxicity even at the higher concentrations of polymers and crosslinker. However, the cells treated with Triton-X 100 as positive control were killed, which indicates the toxic nature of the compound. This shows that the Ge/Pec hydrogels are biocompatible with no detectable toxicity, and can be utilized as a safe carrier for controlled drug delivery.^52-56

Fig. 19. In vitro cytocompatibilty of Ge/Pec hydrogel in the Vero cell line (African green monkey cell line). Data show the mean ± standard deviation of (n=3) independent experiments. Comparison of control and experimental groups for statistical significance was performed with one-way ANOVA. The data was found statistically significant with *P < 0.01.

Discussion

Stimuli-responsive hydrogels have gained increased scientists interactions due to their tremendous response to the diverse external and internal stimuli. The pH is an important stimulus, to which many works have been performed to target this stimulus because the pH of the physiological system changes from a highly acidic condition (stomach) to an almost neutral environment (intestine). The biocompatibility of the materials used in designing such formulations play an important role and should be taken into consideration. In this study, new pH-responsive hydrogels based on Ge/Pec were successfully synthesized and evaluated in details with respect to their physicochemical characterization. Mannitol, an antiasthmatic agent was loaded as a model drug in these Ge/Pec copolymer hydrogels. Ges and Pec belong to natural sources with no toxicity and that’s why are selected for the design of these pH-responsive hydrogels. In our study, we found that Ge/Pec hydrogel has pH-dependent behavior. Given that the Ge/Pec hydrogel contain both amino (NH₂) and carboxylic acid (COOH) groups, when the pH of the solution is above the pKa of an acidic component and below the pKb of a basic component, it results in ionization (and protonation) of acidic and basic components, respectively. The maximum swelling was observed in the buffer solution of pH 1.2 due to the presence of -NH₂ group. In fact, at pH below pKb of basic components, ionization (protonation) occurs resulting in the formation of –NH₃⁺ ions. These ions show electrostatic repulsion due to the presence of similar cations, resulting in the chain repulsion and in turn swelling. Some swelling for Ge/Pec based hydrogels were also observed in the phosphate buffer solution (pH=7.4), which could be due to the presence of carboxylic acid (COOH) groups that result in a deprotonation at higher pH and swelling. Table 2 indicates the effect of pH on dynamic and equilibrium swelling ratios.

The effect of pH on drug release behavior was investigated by immersing the mannitol loaded samples in solutions of different pH (pH 1.2, pH 5.5, and pH 7.5). It was observed that the maximum release occurs at the pH 1.2 (Table 3). It can be correlated with the swelling behavior of the Ge/Pec hydrogel samples, where the maximum swelling was found at the acidic pH. In this study, the effects of different ratios of polymeric agents and crosslinking agent have been investigated.

Three formulations of Ge/Pec hydrogels with varying concentrations of Ge 12.8 g, 14.4 g and 16.0 g were synthesized and subjected to the swelling and release study in the buffer solutions of variable pHs. The swelling coefficients of prepared hydrogels were found to be increased with the increase in the concentration of Ge. At the pH below the pKb value, the Ge chains remain deprotonated. As a result, the chains contain NH₃⁺ ions, and the cationic repulsion between them could be responsible for their high swelling. Same results were found by Saarai et al, who synthesized Ge/Na-Alg hydrogels. Release studies of mannitol were also conducted for samples with increasing concentrations of Ge, in solutions of variable pH values (pH 1.2, 5.5 and 7.5). It was observed that with the increase in the concentration of Ge drug release was also increased at lower pH value. This is attributed to be due to the increased protonation of amino groups at lower pH values. The effect of the concentration of Ge on the release of mannitol from the loaded hydrogel samples was studied (Fig. 7). Moreover, the effect of variable contents of Pec in feed composition of hydrogels was also investigated. Three Ge/Pec hydrogel formulations with varying concentration of Pec (0.9 g, 1.06 g and 1.20 g) were synthesized. It was observed that at low pH values, swelling ratio with increased Pec concentration is not significant as compared to the swelling ratio at high pH values. This is because at low pH values carboxyl groups (-COOH) of Pec remains protonated and same ions repulsive force is eliminated as a result swelling ratio remains low. At pH values above 4, the -COOH group is ionized (deprotonated) and electrostatic repulsion of –COO-(anion-anion repulsion) causes the enhancement of swelling. The swelling and drug release behavior of Ge/Pec hydrogels were also found to be dependent on the contents of crosslinking agent (GA). In order to investigate the effect of GA on swelling and release behavior of hydro gels, a series of three Ge/Pec hydrogels with different GA concentrations (3.8, 4.2 and 4.6% of Ge and Pec) were prepared (Table 2 and Table 3). It was found that the gel swelling was decreased with the increase in GA concentration owing to the presence of physical entanglements in the hydrogel's structure. Further, at the higher concentration of crosslinker, the polymer chain relaxation was decreased, resulting in the less swelling of hydrogels. Similarly, a decrease trend in the swelling ratio has previously been reported by Park et al.

To observe the impact of different concentrations of crosslinking agent, drug release studies were conducted using the buffer solutions at a pH range of 1.2, 5.5 and 7.5. These analyses revealed that the increase in GA contents could result in the reduction in drug release due to the presence of compact structure of hydrogels (Table 3). The sol-gel analysis was also performed for the Ge/Pec hydrogel, and it was found that the gel-fraction of the hydrogel increased along with increased concentration of Ge, Pec and GA. It was also found that the porosity increases by increasing of the contents of Ge and Pec due to increasing of viscosity of the hydrogel solution. Increasing concentration of GA resulted in the decreased porosity, in large part due to the increased physical entanglement between Ge and Pec. The diffusion coefficient of Ge/Pec hydrogel was also calculated, and it was found that the diffusion coefficient reduced with the increasing of GA concentration. Further, it was observed that an increase in the content of Ge resulted in increased values of molecular weight between crosslinks (M_c). Solvent interaction parameter (χ) was studied with results showing that the higher the value of (χ) is, the weaker the interaction between the solvent and polymer will be. Drug release kinetics suggested that the release of drug from the samples with varying Ge compositions and degree of crosslinking follows the zero-order kinetics. The MTT assay showed that there was no significant difference for the cell viability between the treated cell with the hydrogel and untreated control cells. In fact, the hydrogel samples did not show any toxicity even at the higher concentrations of the polymer and crosslinker, indicating that the Ge/Pec hydrogel is biocompatible. The FTIR analysis confirmed the formation of Ge/Pec network and showed that there is no interaction of drug with the materials used. The XRD analysis revealed the crystalline nature of the hydrogels with no chemical interaction between the drug and the hydrogel. The DSC analysis showed the thermal stability of the Ge/Pec hydrogel. The SEM micrographs displayed that the porous nature of the hydrogels facilitates the diffusion of solvent and drug particles into the inter-penetrating network of the hydrogel.

Conclusion

This study presents new pH-sensitive chemically crosslinked hydrogel formulations based on Ge/Pec as a carrier for controlled delivery of antiasthmatic drugs (e.g., mannitol). The swelling of the Ge/Pec hydrogels was affected by the composition and the pH of swelling medium. Swelling studies were conducted in buffer solutions with pH values of 1.2, 5.5 and 7.5. The swelling ratio of Ge/Pec hydrogels showed a regular variation with changing concentrations of Ge, Pec and GA. The swelling ratios were found to be increased by the increase in the concentrations of Ge and Pec, while by increasing the GA concentration, the swelling ratio of the hydrogels were found to be decreased. The drug release from the hydrogel samples was dependent upon the composition of the Ge/Pec hydrogel and the pH of swelling medium. In buffer solution of pH 1.2, the drug release rate was faster than other pH solutions. Non-Fickian release mechanism was found for all the hydrogels. The FTIR spectroscopy confirmed the presence of the essential functional groups and the formation of the crosslinked network. The XRD study indicated that the addition of Pec could reduce the intensity of crystallinity of the Ge/Pec hydrogel. The DSC study showed the thermal stability of the prepared hydrogel samples which had porous structure.

From the pH-dependent swelling and release properties of the Ge/Pec hydrogels, it was concluded that the developed Ge/Pec based hydrogels have pH-sensitive properties with versatile response to the variable environmental conditions. Therefore, these hydrogels can be used as site-specific drug delivery system for drugs with variable solubility.

Ethical approval

There is none to be declared.

Conflict of interests

No potential conflict of interest was reported by the authors.

Acknowledgments

The authors highly acknowledge the Department of Pharmacy, Bahauddin Zakariya University Multan for providing us the laboratory facilities and financing this study.

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