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. 2020 Aug 19;5(34):21610–21622. doi: 10.1021/acsomega.0c02287

Soy Protein-Based Hydrogel under Microwave-Induced Grafting of Acrylic Acid and 4-(4-Hydroxyphenyl)butanoic Acid: A Potential Vehicle for Controlled Drug Delivery in Oral Cavity Bacterial Infections

Saloni Mehra †,‡, Safiya Nisar †, Sonal Chauhan †, Virender Singh §, Sunita Rattan †,*
PMCID: PMC7469417  PMID: 32905438

Abstract

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The objective of this work was to evaluate grafted soy protein isolate (SPI) for pharmaceutical applications. The present work reports the microwave-assisted preparation of soy protein isolate\grafted[acrylic acid-co-4-(4-hydroxyphenyl)butanoic acid] [SPI-g-(AA-co-HPBA)] hydrogel via graft copolymerization using N,N-methylene-bis-acrylamide and potassium persulphate as the cross-linker and initiator, respectively. The chemical and physical properties of the synthesized polymeric hydrogels were analyzed by Fourier transform infrared spectroscopy, liquid chromatography–mass spectrometry (LCMS), nuclear magnetic resonance 1H-NMR, X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The SEM, TEM, and XRD analyses have confirmed the formation of hydrogel SPI-g-(AA-co-HPBA) with the network structure having a layered and crystalline surface. The SPI-g-(AA-co-HPBA) hydrogel was investigated for the sustained and controlled drug delivery system for the release of model drug ciprofloxacin at basic pH for its utilization against bacterial infection in oral cavity. The drug release profile for SPI-g-(AA-co-HPBA) hydrogels was studied using LCMS at the ppb level at pH = 7.4. The synthesized hydrogel was found to be noncytotoxic, polycrystalline in nature with a network structure having good porosity, increased thermal stability, and pH-responsive behavior. The hydrogel has potential to be used as the vehicle for controlled drug delivery in oral cavity bacterial infections.

Introduction

Bacterial infection in oral cavity has been reported to be a serious problem in immuno-suppressed patients because of the colonization of oral cavity by a wide variety of microorganisms.1−3 More than 300 bacterial species have been identified in the periodontal pockets of an adult human.4 Till date, a great number of local drug delivery systems and devices have been proposed for oral applications, among which mucoadhesive drug delivery systems have been reported to show promising effects.5−12 The various advantages associated with mucoadhesive drugs, like prolongation of the residence time of the drug at the site of application, have made the oral drug delivery system as the preferred route of drug administration. These systems remain in close contact with the absorptive tissue, the mucous membrane, and thus contribute to improved and/or better therapeutic performance of the drug for local and systemic effects.13,14 In the last few decades, hydrogels have been explored for their tendency to swell, absorb, and retain a significant amount of water without getting dissolved in it under different biological and biomedical applications.16−21 Although a lot of work has been reported on the use of synthetic hydrogels22 as drug delivery vehicles, however, the use of synthetic hydrogels has been restricted because of many limitations associated with them, such as limited degradability, decreased biocompatibility, and high toxicity. Hence, in the recent past, there has been an increase in the interest among scientists to explore natural polymers as promising candidates for efficient drug delivery systems because of their inherent properties such as biocompatibility23 and biodegradability.

Among the available potential biopolymer drug delivery systems,24,25 protein-based polymers may serve as potential drug delivery agents because of their low cytotoxicity, good biodegradability,26 abundance in nature, high drug binding capacity, and significant uptake into the targeted cells. Moreover, the unique protein structure also provides the possibilities for surface modifications owing to the presence of multiple functional groups in the primary sequences of polypeptides.

In addition, proteins get easily metabolized by digestive enzymes and may generate bioactive peptides that may further exert several physiological effects in vivo.27

Soy protein,28 the most abundant source of plant protein, has gained considerable interest as a greener, renewable, environment friendly, biocompatible, noncytotoxic, and biodegradable material to be explored as efficient drug delivery systems. Soy protein derivatives being the component of functional foods also have a significant role in different biological processes related to the healthy state and prevention of disease. They have been shown to exhibit considerable anticarcinogenic activity, anticholesterolic effect, and protective against obesity and kidney diseases (FDA, 1999).29 The enriched form of soy protein (soy protein isolate, SPI) has been reported to possess high nutritional value and its application in various food items as functional ingredients.

SPI comprises an amphiphilic biopolymer with embedded active functional side chains, which can combine with various reagents.30 The globular structure of this protein comprises two major subunits, conglycinin and glycinin, which contain various amino acids particularly glutamate, aspartate, and leucine characterized by numerous reactive groups that can be used as active sites31 for chemical modifications and cross-linking to develop polymeric hydrogels for biomedical applications.32 SPI, being the major coproduct of soybean oil, is one of the cheapest proteins existing in nature33 with excellent processability34−37 and hence has a promising potential to be developed into a cost-effective hydrogel for industry-scale production. Given its low cost, emulsifying properties, gelling capacity, and nutritional and technological properties, SPI is one of the most frequently studied plant proteins and is already widely used as a carrier for food and nutraceutical applications.38−42 However, their brittle nature, poor physicomechanical strength, water-holding capacity, and moisture sensitivity have limited their applications.43 Only a few studies have focused on their use for biomedical applications44 and its chemical modifications to modulate its functional properties.45

The primary objective of this research was to develop an oral-controlled release drug delivery system based on SPI through grafting of monomers for common antibiotics intended for local treatment of bacterial infections. This property has widely been used to develop polymeric dosage forms for buccal, oral, nasal, ocular, and vaginal drug delivery.46

Attempts were made to design the pH-responsive hydrogel through microwave (MW) irradiation by grafting acrylic acid (AA) and 4-(4-hydroxy phenyl)butanoic acid (HPBA) onto SPI using N,N-methylene-bis-acrylamide (MBA) and potassium persulphate the as cross-linker and initiator, respectively. Graft polymerization of AA endows soy protein films with good tensile properties47,48 as well as modulating its pH response. Further, the inclusion of HPBA along with AA provided mucoadhesive properties,15 in addition to enhancing the swelling characteristics of the prepared hydrogel, leading to the development of an efficient drug delivery system for oral cavity.

The SPI-g-(AA-co-HPBA) hydrogel was investigated for the sustained and targeted controlled drug delivery system49 for the release of model drug ciprofloxacin at basic pH for utilization against bacterial infection in oral cavity. For the first time, the drug release profile for the SPI-g-(AA-co-HPBA) hydrogel has been studied using liquid chromatography LC–MS/MS at the ppb level at pH = 7.4. As LC–MS is highly specific in quantitation using multiple reaction monitoring mode (MRM), which avoids false positive results and can sometime be difficult to rule out using UV technique. In addition, the liquid chromatography mass spectrometer being a highly sensitive instrument can easily detect at ppb (ng/mL) concentrations, which may not be achievable using UV technique.

Results and Discussion

Schematic Representation of Grafting

The graft copolymerization of AA and HPBA on preheated and sodium bisulphite-treated SPI proceeded in three steps: chain initiation, propagation, and termination. The detailed mechanism of graft copolymerization on the backbone of SPI proceeded with the formation of free radical with the K2S2O8 as the initiator, as shown in Scheme 1. The formation of free radical resulted in chain initiation, propagation, and termination reaction52,55,59,65 (Scheme 1). The K2S2O8 generates the radical, which reacts with water to give the hydroxyl radical, it attacks the functional groups on soy protein and initiates grafting copolymerization of SPI, AA, and HPBA, and the free macroradicals are added to double bonds of the monomers, which forms the covalent bond. The radical formation occurs individually simultaneously either on the soy backbone or on the monomer to be grafted first. The mechanism of copolymerization grafting was supported by experimental results obtained by varying different parameters. The grafting with AA and HPBA has resulted in copolymerization and homopolymerization also. The homopolymers formed are removed with excessive washing with protic solvents. Figure S1 represents the images for the experiment in MW.

Scheme 1. Mechanism of Grafting Using Microwave Conditions.

Scheme 1

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Determination of Optimum Grafting Conditions

To obtain maximum grafting percentage (GP%), different reaction parameters such as temperature, preheating temperature, and initiator and monomer concentration were studied under microwave conditions. The results are depicted in Figure 1.

Figure 1.

Figure 1

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(a) Effect of heating temperature in MW on grafting, (b) effect of preheating time in MW on grafting, (c) effect of concentration of initiator on grafting, and (d) effect of monomer concentration on grafting.

Effect of Temperature

The effect of temperature on the graft copolymerization was investigated over the range of 40–100 °C, and the results are shown in Figure 1a. It was observed that GP% increased up to 80 °C temperature, but beyond 80 °C, the GP% started decreasing. These results were similar to other study, which also reported that at low temperature, the redox reaction between potassium persulphate and SPI was slow; however, at elevated temperature, redox reaction became easier and copolymerization GP% increased.52,67 The GP% decreased beyond optimum temperature (80 °C) because termination reaction between radicals and homo polymerization was enhanced.

Effect of Preheating Temperature

The effect of preheating temperature on the graft copolymerization was investigated over the range of 5–20 min under MW, and the results are depicted in Figure 1b. Overall yield of grafting was improved by performing reaction under microwave conditions as per the works reported earlier.53,66 The GP% increased when SPI was preheated to 15 min, and beyond this, the GP% decreases. The preheating of neat SPI at basic pH 8–9 under MW irradiation at high power led to disaggregation of its aggregates and exposed the hydrophobic core of SPI for better polymerization. Preheating results in enhanced crystalline nature of the hydrogel along with increase in porosity,29 as evident from transmission electron microscopy (TEM) images. The scanning electron microscopy (SEM) images for different preheating times are given in Figure S2.

Effect of Amount of Initiator

Grafting was carried out at varying amounts of initiator ranging from 0.01 to 0.10 equiv, and the results, as displayed in Figure 1c, showed that the grafting increased with increment in the amount of initiator up to a certain level, reaching a maximum value (0.05 equiv) and then decreasing sharply.52 With increased amount of initiator, there was an increase in free radicals in the reaction system increasing the GP%. However, with further increase in the amount of initiator, the excessive free radicals increased the tendency of monomers to form homo polymer chains, and termination of the growing chains resulted in decrease in GP%.

Effect of Monomer Concentration

Grafting was carried out at varied monomers concentration. Figure 1d shows that the grafting increased with increase in monomer concentration up to a certain level, reaching a maximum value at optimum concentration of monomers (2.50 g) [SP/AA/HPBA (1:1:1.5)] and then decreasing sharply. The initial increased in GP% was due to the fact that most of the AA and HPBA were utilized by the available free radical sites on the SPI backbone. At higher concentration of monomers, degree of homopolymerization increased and the GP% decreased. Moreover, at lower concentration, the extent of homopolymerization of the monomer was smaller. Thus, grafting reached a maximum value and thereafter decreased, as the number of free radical sites available on the SPI backbone became a limiting factor and thus results in homopolymer formation beyond an optimum value.53

Swelling Behavior

The swelling curves, as displayed in Figure 2, show the swelling behavior of SPI, SPI-g-AA, and SPI-g-(AA-co-HPBA). Neat SPI is the ampholytic polymer, and pH seems to be the limiting factor for affecting the association and dissociation behaviors of SPI in aqueous solution. With the change in pH, the SPI has shown many modifications at the quaternary or tertiary level.29,54 As neat SPI was a globular protein and its hydrophilic core was not exposed to the solvent, therefore, it showed slow swelling at both the pH values, that is, 1.2 and 7.4. In the solution pH of 1.2, the swelling ratio of the both SPI-g-AA and SPI-g-(AA-co-HPBA) hydrogels also exhibited low swelling behavior, as diffusion of water is not facilitated at this pH. At pH below the isoelectric point (pH 4.5) of SPI, the peptide chains of SPI, hydrophilic groups COOH and OH of AA, and HPBA chains attached to the backbone of SPI carried net positive charge, which reduce the association of peptide chains as well as deprotonation of COOH and therefore resulted in reduced hydrophilicity and hence very less swelling ratio at acidic pH (1.2).53 However, SPI-g-AA is a cross-linked polymer network, and the presence of acidic groups showed a faster swelling rate and higher percentage of swelling in basic medium as compared to acidic pH. The pH-responsive acidic groups of the SPI-g-AA hydrogel facilitated the diffusion of water into the hydrogel network.56 The increase in the swelling ratio at basic pH was attributed to highly ionizable groups, which lead to hydrophobic and hydrophilic transition on the SPI backbone. The gradual ionization of AA units at pH higher than its pKa has resulted in increased swelling at pH 7.4. The rate of water adsorption by SPI-g-AA hydrogel increased sharply and then reached an equilibrium state at ∼5 h. The SPI-g-(AA-co-HPBA) exhibited gradual swelling in basic pH, reaching equilibrium at around double the time of ∼10 h. The gradual swelling can be explained because of the presence of both hydrophilic acidic groups and hydrophobic alkyl and aryl chains. The hydrogel demonstrated increased swelling ratio with time, which was the main feature needed for sustained release of drug. The network structure and the cross-linking density have increased because of the introduction of HPBA and AA on SPI as confirmed by SEM, which has shown increase in the cross-linking groups in the SPI-g-(AA-co-HPBA) hydrogel. The overall reduced swelling ratio as compared to SPI-g-AA was attributed to overstock cross-linking density, which has influenced the swelling ratio.53 The gradual swelling capacity in alkaline media made the synthesized hydrogels significantly important in the targeted and controlled drug delivery system in pH 7.4 of oral cavity. The above observations were strengthened by the topology of SEM and TEM. SPI-g-(AA-co-HPBA) clearly indicated the formation of layered polycrystalline nature, which resulted in slow diffusion of water in pH 7.4 between the layers and resulted in gradual swelling for SPI-g-(AA-co-HPBA) as compared to swelling in SPI-g-AA at pH 7.4. SPI-g-(AA-co-HPBA) showed normal Fickian diffusion as (n) approached 0.5 value in basic pH as compared to other. The R2, (n), and (k) for all the three polymers at two different pH values are depicted in Table 1. The values indicated the non-Fickian diffusion behavior for SPI-g-AA and neat SPI.

Figure 2.

Figure 2

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Graph showing% of swelling behavior of (a) (1) SPI-g-(AA-co-HPBA), (2) neat SPI, and (3) SPI-g-AA at pH 7.4 and (b) (1) neat SPI, (2) SPI-g-AA, and (3) SPI-g-(AA-co-HPBA) at pH 1.2.

Table 1. Showing R2, n, and k Parameter to Identify Swelling Kinetics of Neat SPI, SPI-g-AA, and SPI-g(AA-co-HPBA) at pH 1.2 and pH 7.4.

sample name pH R2 N k
neat SPI pH 1.2 0.802 0.344 2.11
SPI-g-AA pH 1.2 0.705 0.352 2.14
SPI-g-AAHPBA pH 1.2 0.911 0.372 2.3
neat SPI pH 7.4 0.835 0.353 2.1
SPI-g-AA pH 7.4 0.761 0.222 1.3
SPI-g-AAHPBA pH 7.4 0.971 0.425 2.7
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Experiment of performing swelling study at 37 °C is illustrated in Figure S3.

Drug Absorption and Release Study

Drug Absorption Study

This study was performed using the Agilent 6470 LC–MS/MS instrument, where two MRM transitions 332.1/314.1 and 332.1/231 were used for monitoring the quantitation of ciprofloxacin. Linearity of ciprofloxacin was established from 5 to 1000 ppb with coefficient of regression R2 > 0.997, as shown in Figure 3a. Ciprofloxacin (660 mg) was dissolved in 820 mL of methanol and 300 mL of Milli-Q water. The mixture was heated at 60–80 °C for 2 h. After clear solution was formed, 305 mg of SPI-g-(AA-co-HPBA) hydrogel was added to the above solution, and readings were taken for 20 h. The absorption study was investigated with LC–MS using the Agilent 6470 Triple Quadrupole LC/MS system, as shown in Figure 3b. It was observed that the rate of absorption of ciprofloxacin on the SPI-g-(AA-co-HPBA) hydrogel remained high for 7–8 h, and thereafter, it showed slow absorption, and finally after 10 h, it became saturated. Figure 3b represents the absorption of ciprofloxacin onto the SPI-g-(AA-co-HPBA). It was evaluated from the graph that more than 50% of the drug got loaded onto the hydrogel as it has good porosity and pore size.

Figure 3.

Figure 3

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(a) Calibration curve for linearity of ciprofloxacin. (b) Absorption study of ciprofloxacin on the hydrogel by LC–MS/MS.

Kinetics and Mechanism of Drug Release

The kinetics of drug release was studied by employing zero order and first order kinetic models. The mechanism of drug release was evaluated by using the same Ritger–Peppas model,57 which can be defined as follows for drug release

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where Mt and Meq are the drug released at specific time t and equilibrium, respectively. (n) is the diffusion coefficient, and (k) is the constant depending on geometrical and structural characteristic of the hydrogel. The drug delivery capability of the fabricated hydrogels was evaluated using ciprofloxacin as the model drug for in vitro release. The in vitro release results show that the amount of ciprofloxacin released was characterized by slow and sustained release at the physiological pH 7.4, as expected. As shown in Figure 4a,b, the hydrogels demonstrated gradual drug release till 14 h, upto which the study was carried out.56 The gradual drug release was attributed to the presence of hydrophobic and hydrophilic centers in SPI-g-(AA-co-HPBA). The increased cross-linking density because of the introduction of AA and HPBA has enhanced the drug loading of SPI. The controlled swelling has increased the bioavailability of drug for oral infections over the prolonged time. As the value of (n) was less than 0.5, it reflected non-Fickian diffusion release of the drug from the matrix, as shown in Table 2. The hydrogel was observed to be stable at physiological pH but was cleaved at acidic pH.

Figure 4.

Figure 4

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(a) Ciprofloxacin release study from the grafted hydrogel at pH 7.4, and (b) percentage release of ciprofloxacin from the grafted hydrogel at pH 7.4 over 14 h, using Agilent 6470 LC–MS/MS.

Table 2. Study of Kinetics of Drug Absorption at pH 7.4.
study of ciprofloxacin released R2 n k
drug release study at pH 7.4 0.9888 0.311 2.063
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The drug release in acidic medium was also studied and is given in Figure S4.

Crystallographic Studies (XRD)

The diffraction pattern of neat SPI and SPI-g-(AA-co-HPBA) is depicted in Figure 5, which showed a broad band at 2θ value around 20°, which was the characteristic peak for pure SPI globular 7s and 11s amorphous components and corresponds to the amorphous nature of neat SPI having low tensile strength. The graft copolymerization of AA and HPBA resulted in an increased crystallinity of SPI as compared to amorphous nature of neat SPI.58 The X-ray diffraction (XRD) pattern for grafted soy protein, as represented in Figure 5, showed characteristic peaks at 2θ values around 18 and 19, 21 and 25, 31 and 33, 38 and 39, and 58 and 59° corresponding to the isomer of SPI. These peaks correspond to different facets formed because of the introduction of crystallinity in hydrogel.64 The neat SPI was a globular aggregated protein, and its disulphide bonds were broken using sodium bisulphite during preheating, which opened the backbone of globular proteins for grafting. The randomly arranged protein matrix became organized to form well distributed polycrystalline and mesophase and this resulted in the increase in crystallinity. The increase in the degree of crystallinity directly increased the tensile strength of the polymer. The formation of polycrystalline natured hydrogel was confirmed with symmetrical dot circle in TEM images for grafted SPI.

Figure 5.

Figure 5

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XRD data for neat SPI and SPI-g-(AA-co-HPBA).

Fourier Transform Infrared Spectroscopy

The Fourier transform infrared spectroscopy (FTIR) peak of neat SPI, as shown in Figure 6, displayed a strong band at 3463 cm–1, which was because of the mixed absorption of N–H and O–H stretching. The second characteristic peak of neat SPI showed two bands at 1651 and 1534 cm–1 for amide-I and amide-II bonds,55−59 respectively, which showed that N–H and C–O were in the trans position. It showed weak bands at 1457 cm–1 for deformation of C–H and 669 cm–1 corresponds to out-of-plane bending of O–H. The characteristic bending of globular SPI because of S–H appeared at 2550 and C–S at 1050 cm–1. The grafted SPI-g-(AA-co-HPBA) has shown significant changes in the FTIR as compared to neat SPI. The broad peak at 3293 cm–1 indicated the change in O–H and N–H stretching in grafted SPI and indicated that the OH group of HPBA was also involved in hydrogen bonding with SPI. Medium and intense peaks at 1650 and 1180 cm–1 correspond to carbonyl stretching and C–O stretching of AA and butanoic acid, respectively. The disappearance of bands in grafted SPI at 2550 and 1050 cm–1 indicated breaking of disulphide bonds during preheating under MW, which further confirmed the disaggregation of globular SPI. The strong peak at 618 cm–1 indicated the free SH bond after breaking of disulphide bonds.52 It was observed from Figures 6, S5 and S6 that there was a change of intensity of peaks with different grafting percentages while performing experiments for optimum conditions. It was well established that when graft copolymerization takes place, the macromolecular end groups are redistributed, which result in the change of intensity of peaks in FTIR. Therefore, grafting on the backbone of SPI with AA and HPBA was well established by the changes observed in FTIR.

Figure 6.

Figure 6

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FTIR spectra of neat SPI and SPI-g-(AA-co-HPBA).

Thermogravimetric Analysis

Thermal stability of SPI and SPI-g-(AA-co-HPBA) hydrogel was investigated using thermogravimetric analysis (TGA), in which weight loss was recorded within 100–900 °C, as shown in Figure 7. In case of neat SPI,58,60 the first 7–8% weight loss of soy protein occurred because of elimination of water and breaking of its globular aggregate. After this, neat SPI showed 10–12% weight loss in the range of 280–290 °C and 12–34% weight loss occurred at 300–400 °C because of degradation of covalent bonds, dissociation of 7s and 11s subunits, and cleavage of peptide bonds of amino acid. Secondary protein structures were reassociated because of hydrophobic and electrostatic interactions with the disulphide bond. Upon further heating upto 800–900 °C, up to 64% of weight loss occurred because of complete degradation and liberation of various gases and leaving 36% residue because of the formation of carbon ashes. In case of grafted SPI-g-(AA-co-HPBA), only 2–3% loss occurred because of elimination of water upto 100–110 °C. Grafted SPI clearly showed that the first weight loss of 10–15% occurred because of breaking of covalent bonds at sharp 315 °C. It clearly marked the increased polycrystallinity of grafted SPI as compared to neat SPI, which results in the enhanced thermal stability. The second decomposition of around 25–40% was attributed to the decomposition of proteins; loss because of release of CO2 and other gases at 500–600 °C and the degradation of grafted chains. Because of the increase in thermal stability, the grafted SPI exhibited only 42% decomposition as compared to 64% decomposition of neat SPI. Further, 57% residue was left after heating at 800–900 °C because of the increase in carbon content in grafted SPI.

Figure 7.

Figure 7

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TGA/DTG of neat SPI and SPI-g-(AA-co-HPBA).

Scanning Electron Microscopy

The topology of neat SPI, SPI-g-AA, and SPI-g-(AA-co-HPBA) was analyzed by using SEM, as represented in Figure 8. Figure 8a shows neat SPI as globular-shaped protein with a granular morphology. The neat SPI was neither uniform in size nor in shape.58,59 It indicated the aggregated form of neat SPI, where no active sites were exposed. Figure 8b–h shows sharp change in the morphology of grafted SPI-g-AA and SPI-g-(AA-co-HPBA). The preheating in MW and sodium bisulphite resulted in disaggregation of the globular protein, and hence, the free NH2 and COOH groups on the SPI backbone were available for grafting with AA and HPBA, respectively. SPI-g-AA, as shown in Figure 8b–d, exhibited a coarse surface morphology. The size and shape of the particles were not uniform. The surface, as shown in Figure 8c, has shown the amorphous nature of graft. The topology, as shown in Figure 8d, depicted that porosity has increased, and whole surface has grafted properly and hence the swelling ratio as compared to neat SPI. Figure 8e–h depicts the surface morphology for SPI-g-(AA-g-HPBA) hydrogel; the newly formed hydrogel has shown network structures, which have a layered surface. Figure 8e displays self-assembly of the unfold peptides from SPI. The extended surface (small villi type extension) has resulted in the increase in porosity as compared to neat SPI. The heterogeneity on the surface and cavities formed, as shown in Figure 8f, resulted in good loading of drug in these cavities. The presence of free OH and COOH groups on HPBA resulted in the formation of covalent bonds and hydrogen bonds with the free NH2 and COOH groups of the SPI, which resulted in the formation of peptide bonds53 and hence uniformed the layer, as depicted in Figure 8h. The layered structure will further help in film forming for mucoadhesive patches as phenol and acid groups introduced increases the plasticizing property of SPI films. To overcome the brittleness for mucoadhesive SPI films, HPBA was added along with AA. As per the literature, introduction of phenolic groups like catechol and ferulic acid largely increased the plasticizing property of SPI films by increasing the cross-linking in the protein matrix.15,59

Figure 8.

Figure 8

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(a) SEM of neat SPI (×3.0 K) at 10 μm, (b) SPI-g-AA (×20.32 K) at 1 μm, (c) SPI-g-AA (×30.8 K) at 1 μm, (d) SPI-g-AA (×8.4 K) at 2 μm, (e) SPI-g-(AA-co-HPBA) (×2.07 K) at 2 μm, (f) SPI-g-(AA-co-HPBA) (×30.84 K) at 1 μm, (g) SPI-g-(AA-co-HPBA) (×20.0 K) at 2 μm, and (h) SPI-g-(AA-co-HPBA) (×8.15 K) at 1 μm.

Transmission Electron Microscopy

TEM images, as shown in Figure 9, clearly depicted the size, shape, and size distribution of particles in neat SPI and grafted SPI.61Figure 9a,b, indicates very less particle size and less porous structure of neat SPI. Figure 9c represents the globular and amorphous structure of protein. Figure 9d shows that the electron image of neat SPI was globular in nature and folded proteins. Figure 9e explains the increased porosity of grafted SPI-g-(AA-co-HPBA) as the size of pores increased to 21–30 nm. The empty spaces were occupied by monomers of AA and HPBA. Figure 9f shows equal-sized particles in layers. The formation of covalent bonds between free COOH and OH of HPBA with the SPI backbone to form fixed peptide bonds, as depicted in Figure 9g, which clearly showed the series of ring of dots, indicating the polycrystalline diffraction pattern. The distance between the layers is also calculated. Figure 9h represents the electron image of SPI-g-(AA-co-HPBA), which has depicted the formation of network structures and complete change in the bonding of protein. Grafting SPI with AA and HPBA formed a polycrystalline-layered structure, resulting in the controlled swelling behavior.

Figure 9.

Figure 9

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TEM images: (a) neat SPI at 50 nm, (b) neat SPI at 100 nm, (c) neat SPI at 51 nm, (d) neat SPI electron image at 5 μm, (e) SPI-g-(AA-co-HPBA) at 50 nm, (f) SPI-g-(AA-co-HPBA) at 1 μm, (g) SPI-g-(AA-co-HPBA) at 51 nm, and (h) SPI-g-(AA-co-HPBA) electron image at 1 μm.

Nuclear Magnetic Resonance (1H NMR)

The chemical structure of neat SPI and grafted SPI-g-(AA-co-HPBA), as shown in Figure 10, was characterized by 1H-NMR by dissolving the SPI and SPI-g-(AA-co-HPBA) in D2O by adjusting pH upto 9.0 with crushed sodium hydroxide.56 An aromatic region showed the signal 7.5–6.5 ppm because of the amide group of different amino acids present in SPI. In grafted SPI, intergration increased because of the presence of phenyl group of HPBA. As there was no signal in the region of 5–5.5 ppm, it clearly marked the absence of monomer of AA in the graft. 1H NMR of grafted SPI clearly showed the triplet of CH2 at 3.71 and 2.34 ppm and mutliplet of CH2 at 1.7–1.67 ppm because of HPBA and CH at 2.64 ppm because of the AA polymer chain. Figure S7 shows overlay 1H NMR of (a) neat SPI (b) SPI-g-(AA-co-HPBA).

Figure 10.

Figure 10

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1H NMR in D2O comparison of neat SPI and SPI-g-(AA-co-HPBA).

Antibacterial Activity (Well Diffusion Assay)

The antimicrobial activity of neat ciprofloxacin, SPI-g-(AA-co-HPBA) hydrogel, and ciprofloxacin-loaded SPI-g-(AA-co-HPBA) hydrogel was evaluated by monitoring the zone of inhibition (ZI) against the growth of Escherichia coli and is displayed in Figure 11. ZI did not appear in the untreated group, which did not receive any treatment in the well formed on the agar plate, and the growth of E. coli was observed on the complete surface. However, neat ciprofloxacin exhibited significant antimicrobial activity as revealed by ZI with a diameter of 2.9 cm. The ciprofloxacin-loaded SPI-g-(AA-co-HPBA) hydrogel revealed an increase in diameter of the (ZI-3.1 cm) in comparison to neat ciprofloxacin (ZI-2.9 cm), although the increase was not significant at P < 0.05. Appreciable antimicrobial activity was also exhibited by SPI-g-(AA-co-HPBA) hydrogel alone (without ciprofloxacin); however, the diameter of ZI for SPI-g-(AA-co-HPBA) hydrogel alone was 0.67 cm, which was significantly smaller than that shown by neat ciprofloxacin (2.9 cm) or ciprofloxacin-loaded SPI-g-(AA-co-HPBA) hydrogel (3.1 cm).

Figure 11.

Figure 11

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Antibacterial activity (ZI size) of neat ciprofloxacin, SPI-g-(AA-co-HPBA) hydrogel, and ciprofloxacin-loaded SPI-g-(AA-co-HPBA) hydrogel against E. coli bacterial strain. The untreated group did not receive any treatment in the well formed on E. coli-plated agar plates.

Cytotoxicity

The cytotoxic activity of different test agents was evaluated by MTT assay in human hepatocarcinoma (HepG2) cells for different doses (40–160 μL each of 1 mg/mL test solution), and the results are displayed in Figure 12. SPI neat represented the neat SPI. CP-loaded hydrogel and SPI-g-(AA-co-HPBA) represented the loaded and unloaded hydrogel. CP neat represented the ciprofloxacin alone, which was used as a model drug. Neat HPBA represented 4(4-hydroxyphenyl)butanoic acid. SPI62,64 (SPI neat) did not affect the viability of the HepG2 cells in comparison to the untreated control (viability 100%) and exhibited a slight decrease in cell viability; however, the percent decrease was not significant at p < 0.05. The cell viability thus remained comparable to the control group (Figure 12). Unloaded hydrogel (SPI-g-AA-HPBA) did not decrease the cell viability significantly at a dose of 40 μL; however, at higher doses of 80, 120, and 160 μL, the viability decreased in a significant manner to 77, 68, and 63%, respectively, in comparison to the control (100%). Neat ciprofloxacin (CP-neat) exhibited maximum toxicity to the HepG2 cells, and the viability reduced significantly at all the tested concentrations with respect to the control (Figure 12). The ciprofloxacin-loaded hydrogel (CP-loaded hydrogel) though exhibited slight toxicity to the cells in a concentration-dependent manner but remained lesser toxic in comparison to CP neat at each tested concentration. CP neat decreased the cell viability within the range of 61–35%; however, the CP-loaded hydrogel decreased the cell viability within the range of only 78–58% (Figure 12). Neat HPBA also showed slight cytotoxicity to the HepG2 cells in a concentration manner and reduced the viability to 84, 74, 71, and 70% at 40, 80, 120, and 160 μL, respectively. Figure S8 shows the MTT assay for cytotoxicity study.

Figure 12.

Figure 12

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Effect of different treatments on the viability of HepG-2 cells as evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The control group was not subjected to any treatment and was considered to have 100% cell viability. The effect of different treatments on cell viability has been presented as per cent of control group (100%). Each value depicts an average of three sets of experiments and was expressed as mean ± SE. Significance at *P < 0.05, compared with the control (Student’s t-test).

Conclusions

The pH-responsive SPI-g(AA-co-HPBA) hydrogels were synthesized successfully for controlled release of drug in oral cavity. Instant absorption of drugs by the mucosa of the oral cavity renders the drug less effective and may also elicit adverse reactions; however, sustained release of drug through hydrogels will make it possible to better define therapeutic clinical protocols for oral infections. By using optimum condition for grafting, we developed the green route to graft the SPI using preheating, sodium bisulphite, AA, HPBA, initiator, and cross-linker. The results indicated the formation of grafted biodegradable polymer, which resulted in more than 50% loading of drug, as studied by LC–MS/MS. The newly formed [SPI-g-(AA-co-HPBA)] hydrogel is having appreciable antimicrobial property, less cytotoxity, polycrystalline nature, network structure, good porosity, increased thermal stability, and pH responsiveness for the delivery of drug in the predetermined rate over the period of 12 h in oral cavity. The SPI-g-(AA-co-HPBA) hydrogel, thus, has considerable potential of sustained and targeted drug delivery and may be used against oral infections.

Experimental

Materials

SPI (Nutrimed Healthcare Private Limited), AA (99%, Fluka), sodium metabisulphite (98%, Sigma-Aldrich), HPBA (99%, Combi blocks), potassium persulphate (98%, Merck), MBA (97%, GLR), and ciprofloxacin hydrochloride salt (97%, TCI) were obtained. All the solvents used were of analytical grade (Merck, Mumbai). The water used to prepare buffers was double-distilled Milli-Q water. MTT and dimethyl sulfoxide (DMSO) were purchased from M/s Qualigens (India). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), trypsin 0.25%, antibiotic solution, trypan blue, nutrient broth, and nutrient agar were procured from M/s HiMedia (India). Ciprofloxacin was used as the model drug for the study of controlled drug release using the SPI-g-(AA-co-HPBA) hydrogel.

Methods

Microwave-Assisted Preparation of AA and 4(4-Hydroxyphenyl)butanoic Acid-Grafted Soy Protein Isolate

To the argon-purged dried microwave vial, 1.00 g of SPI was added at room temperature in 10 mL of double-distilled water (pH-9.0). The suspension was irradiated under Anton Parr microwave at 60 °C for 15 min. The reaction was stopped, and sodium bisulphite (202.0 mg, 1.94 mmol) was added to the above suspension at room temperature under argon purging. The vial was sealed and irradiated in microwave (400 W) at 85 °C for 10 min. The reaction was stopped, and to the above suspension, AA (1.00 g, 13.87 mmol) and HPBA (1.50 g, 8.25 mmol), followed by addition of MBA (75 mg, 0.48 mmol) and potassium persulphate (0.280 g, 1.04 mmol), were added at room temperature under an argon atmosphere. The reaction vial was again purged with argon for 10 min to ensure the removal of oxygen radical. The sealed vial was again irradiated under microwave radiation at 85 °C for 30 min at 400 W. The reaction mixture was quenched with distilled water and filtered. The filtered solid was washed with water, ethanol and, acetone to remove excess of monomer and homomonomers. The resulting hydrogel was dried for 2 days under vacuum.

The percentage of grafting and the degree of grafting was calculated by using the following equation

graphic file with name ao0c02287_m002.jpg 2
graphic file with name ao0c02287_m003.jpg 3

where Wg, Wo, and W1 are weight of the grafted SPI, weight of neat SPI, and weight of the monomer used for grafting, respectively.

Percentage of grafting and grafting efficiency has been determined as a function of amount of initiators, preheating temperature, and amount of monomer concentration and temperature of reaction.

Instruments and Characterization

Crystallographic Studies (XRD)

An XRD model Rigaku Ultima IV X-ray diffractometer operating at 40 KV and 40 mA, λ = 0.154 nm, 2θ = 5°–90°, and step width of 0.02° was used to identify the crystallinity of the newly formed hydrogel.

Fourier-Transform Infrared Spectroscopy

FTIR measurements were performed using the Nicolet 5700 FTIR spectrophotometer in the spectral range from 4000 to 400 cm–1 to confirm the grafting of SPI. Samples were dried completely and ground with potassium bromide, and discs were prepared by compression molding under vacuum.

Thermal Gravimetric Analysis

The thermal stability of the hydrogels was examined using a Q500 Thermal Gravimetric Analyzer (TGA). All vacuum-dried samples were heated from room temperature to 900 °C under a N2 atmosphere at a heating rate of 10 °C. The TGA tests were performed under an inert atmosphere, so that the SPI transformed into carbon at high temperatures instead of going through complete oxidation.

Scanning Electron Microscopy

The surface morphologies of pristine SPI and SPI-g-(AA-co-HPBA) hydrogel were investigated using Leo 435 V P SEM at different resolutions. The samples were sputter-coated with gold and were examined using SEM.

Transmitting Electron Microscopy Analysis

The distribution morphology of SPI and SPI-g-(AA-co-HPBA) was examined by using JEM-2100F (JEOL, Japan) high-resolution TEM, operated at 200 kV. The ultrathin (∼70 nm) sections of film samples embedded in cured epoxy resin for TEM examination were obtained using a microtome (Leica EM UC7, Germany) at a rate of 10 mm·min–1 and collected with a copper grid. The grids were stained with uranyl acetate (10 min) and washed thrice with deionized water.

Nuclear Magnetic Resonance (1H NMR)

The proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker 400 MHz spectrometer to elucidate the formation of hydrogel having AA and HPBA in D2O by adjusting pH upto 8–9 with crushed sodium hydroxide.

Liquid Chromatography–Mass Spectrometry

LC–MS was performed using the Agilent 6470 Triple Quadrupole LC–MS system to study the absorption and release of the model drug ciprofloxacin on hydrogel.

Equilibrium Swelling Capacity and Swelling Kinetics Measurements

The equilibrium swelling ratio (SR) of hydrogels was investigated at pH 1.2 (gastric fluid) and 7.4 (oral cavity) at 37 °C. The preweighed dried hydrogels (1.0 g) were immersed in phosphoric buffer solution and left to swell. The swollen hydrogels were taken out at regular intervals, and excess water was removed using filter papers and weighed. To ensure complete swelling, the samples were allowed to swell for 12 h. SR was calculated using the following relation

graphic file with name ao0c02287_m004.jpg 4

where Md is the weight of the dry hydrogel, and Mt is the weight of the swollen hydrogel at equilibrium.

To analyze the swelling kinetics, first order and second order kinetic models were used. To analyze the swelling mechanism of the hydrogels, the Ritger–Peppas model was employed, which can be defined by the following equation

graphic file with name ao0c02287_m005.jpg 5

where Mt and Meq are the amount of water absorbed at time t and at equilibrium, respectively, (k) is the characteristic constant of the hydrogel, and (n) is the characteristic exponent of the mode of water transport mechanism. In the graph between log(Mt/Meq) versus log t, the slope and intercept of the plot give the value of (k) and (n), respectively. The value of (n) = 0.5 signifies a normal Fickian diffusion, value of (n) = 1 signifies case II diffusion, and the value of (n) between 0.5 and 1 indicates non-Fickian or anomalous diffusion.

Drug Absorption Study

The drug absorption profile of model drug ciprofloxacin was studied using the Agilent 6470 LC–MS/MS instrument. Ciprofloxacin was dissolved in methanol and Milli-Q water, followed by addition of SPI-g-(AA-co-HPBA) hydrogel to the above solution, and readings were taken for 20 h. The absorption study was investigated with LC–MS using the Agilent 6470 Triple Quadrupole LC/MS system. The stirring was stopped, and the solid was filtered and washed thoroughly with water, ethanol, methanol, and acetone to remove unreacted ciprofloxacin. The drug-loaded hydrogel was dried and used further to study release under basic pH.

Drug Release Study

The release profile of model drug ciprofloxacin63 from the drug-loaded hydrogel was studied at pH 7.4 of oral cavity. The drug-loaded hydrogel (100 mg) was immersed in 100 mL of phosphate buffer solution of pH 7.4, and the release was monitored at 37 °C. Aliquots(1 mL) were withdrawn after every 1 h, and release study was done using LC–MS technique at the ppb level.

Biological Studies

Antimicrobial Behavior

The antibacterial activity of the hydrogel was investigated by well diffusion assay by measuring area of ZI as described by Holder and Boyce (1994).50 Soft nutrient agar plates were prepared using Petri dishes of 90 mm diameter. The agar medium was sterilized by autoclaving it in a conical flask at a pressure of 15 lbs for 30 min. The medium was then transferred into presterilized Petri dishes in a laminar air flow chamber, and dishes were allowed to cool and solidify at room temperature. Thereafter, E. coli (OD600 = 0.6–0.8 ∼ 2 × 108 CFU/mL) was seeded as test organism for antimicrobial assay. The culture was evenly spread on the media with a sterilized glass spreader. Wells of equal size (10 mm in diameter) were bored at the center of each agar plate using a metal borer. Varying concentrations of hydrogel, active monomer, neat SPI, and ciprofloxacin were prepared in PBS (500 μL final volume) and poured in each well. The plates were incubated at 37 °C for 24 h. Thereafter, ZI was measured.

Cytotoxicity Evaluation

The relative cytotoxicity of the hydrogels on HepG2 cell line was evaluated by MTT viability assay as described by Shahneh et al., (2013).51 Briefly, HepG2 cells were plated in triplicate at a density of 5 × 103 cells/well each having 200 μL culture medium (DMEM supplemented with 10% FBS and penicillin/streptomycin, 100 μg/mL) in a 96-well plate and incubated at 5% CO2 in the incubator at 37 °C. After incubation for 24 h, cells were treated with four different concentrations of 40, 80, 120, and 200 μL of 1 mg/mL in 100 μL medium of each neat SPI, neat HPBA, SPI-g-(AA-co-HPBA), and SPI-g-(AA-co-HPBA) + drug and neat ciprofloxacin. Thereafter, the culture plates were incubated in the CO2 incubator for 24 h, and the cells were allowed to proliferate in DMEM containing hydrogel suspensions or PBS. After this, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the cells were incubated at 37 °C for 4 h to allow MTT to be metabolized. Media were then discarded, and the plate was allowed to dry. The resulting formazan crystals in each well were solubilized by the addition of 200 μL DMSO. The absorbance was measured at 570 nm using the micro plate reader (Bio-Rad India). The viability of cells incubated in the medium containing DMEM and PBS was considered to be 100%.

Statistical Analysis

All the data have been presented as mean ± SE. Student’s t-test was applied for determining the statistical significance between different groups.

Acknowledgments

The authors are thankful to Jubilant Chemsys, Noida and Amity University Uttar Pradesh, Noida for providing the neccesary experimental support. The authors sincerely thank the referees for suggesting important modifications to the manuscript.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02287.

  • Experiment performed in Anton Parr MW under argon, 5 min preheating in MW and (b) 15 min preheating in MW, swelling studies performed at 37 °C at pH 7.4 and 1.2, study of drug release at pH 1.2, overlay of effect of preheating in MW for 5 and 10 min on grafting percentage, overlay of effect of monomer concentration on grafting percentage, overlay of 1H NMR of (a) neat SPI and (b) SPI-g-(AA-co-HPBA), and MTT assay for cytotoxicity study (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c02287_si_001.pdf (314.5KB, pdf)

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