Development and Evaluation of the Biological Activities of a Plain Mucoadhesive Hydrogel as a Potential Vehicle for Oral Mucosal Drug Delivery
Abstract
This study aimed to develop HGs based on cationic guar gum (CGG), polyethylene glycol (PEG), propylene glycol (PG), and citric acid (CA) using a 2k factorial experimental design to optimize their properties. HGs were characterized through FTIR and Raman spectroscopy, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The biological activities of HGs were determined by evaluating their mucoadhesive capacity and antibacterial activity in vitro, whereas their toxicity was analyzed using Artemia salina nauplii as an in vivo model. Results revealed that HGs were successfully optimized for their viscosity, pH, and sensory properties, and it was observed that varying concentrations of PEG-75 did not influence them. Through SEM analyses, it was noted that increased levels of PEG-75 resulted in HGs with distinct porosity and textures, whereas FTIR and Raman spectroscopy exhibited representative peaks of the raw materials used during the synthesis process. TGA studies indicated the thermal stability of HGs, as they presented degradation patterns at 100 and 300 °C. The synthesized HGs exhibited similar mucoadhesion kinetic profiles, demonstrating a displacement factor at an equilibrium of 0.57 mm/mg at 5 min. The antibacterial activity of HGs was appraised as poor against Gram-positive and Gram-negative bacteria due to their MIC90 values (>500 μg/mL). Regarding A. salina, treatment with HGs neither decreased their viability nor induced morphological changes. The obtained results suggest the suitability of CGG/PEG HGs for oral mucosa drug delivery and expand the knowledge about their mucoadhesive capacity, antibacterial potential, and in vivo biocompatibility.
Introduction
Oral mucosa performs multiple vital functions for general health and well-being. Anatomically, the oral mucosa consists of the oral epithelium, lamina propria, and submucosa [1]. Physiologically, it acts as a physical and immunological barrier against a wide range of external stimuli, including mechanical, chemical, and biological agents, as well as carcinogenic substances and oral bacteria [2,3]. Additionally, the oral mucosa plays a key role in secretion, housing salivary glands responsible for producing saliva, which maintains tissue moisture, facilitates digestion and lubrication, and plays an immunological role [4].
Saliva is a complex secretion that protects soft tissues, controls dryness, and can influence tissue repair. Composed of 99% water and 1% organic and inorganic molecules, saliva has a pH range between 7 and 7.4 [5]. A persistent decrease in pH can lead to symptoms such as cervical caries, gingival recession, cervical erosion, demineralization, and white spots on enamel [6]. In contrast, mucin, a complex and viscous substance produced and secreted by specialized cells in columnar epithelia, plays a crucial role in protecting underlying epithelial tissues by providing lubrication, hydration, and a barrier against harmful agents and pathogens [7].
Biochemically, mucin is abundant in serine, threonine, and proline, which are aminoacidic residues extensively glycosylated with fucose, galactose, sialic acids, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc) [8,9]. Sulfate ester groups and carboxyl groups of sialic acids in oligosaccharides confer a net negative charge to mucin, varying with oligosaccharide composition [10,11]. This negative charge is crucial for drug formulation in oral mucosa, as it necessitates positively charged carriers for effective adhesion and drug delivery effectiveness [12]. For example, recent reports have documented that positively charged systems such as chitosan nanoparticles, polymethacrylate micelles, and β-cyclodextrin/dialdehyde interact electrostatically with negatively charged mucin, enhancing the retention and bioavailability of therapeutic agents such as bevacizumab, azithromycin, and insulin, respectively [13,14,15].
Hydrogels (HGs) are polymeric materials capable of absorbing and retaining large amounts of water or other fluids [16]. In comparison to other materials, HGs are highly advantageous since they possess properties useful for biomedical applications, such as biocompatibility, biodegradability, stimulus responsiveness, and controlled release of active substances [17]. In addition, they are considered suitable materials for biomedical applications as they can successfully deliver therapeutic agents (e.g., plant extracts, isolated compounds, or peptides) experimentally or clinically [18,19] and pose ease of application through distinct administration routes and versatility to be formulated in a liquid or semi-solid form [20].
Currently, the global market size of HGs is estimated at USD 23.16 billion in 2024, but it is expected to increase to USD 32.62 billion by 2029. The evident economic growth of HGs in current markets can be attributed to their wide range of applications in the healthcare industry, especially for treating oral and maxillofacial diseases [21], periodontitis [22], recurrent aphthous ulcers [23], radiation or chemotherapy-induced oral mucositis [24], and chronic oral lesions of immunological origin [25]. As with other materials, the assembly, size, shape, and surface features of HGs can be controlled by optimizing their manufacturing, and there are various analyses (e.g., density functional theory and molecular dynamics analyses) designed and implemented to fulfill this purpose [26,27]. The factorial regression analysis is an advantageous statistical approach that enables the evaluation of the effect of multiple independent variables on a response variable, and it is highly suitable for identifying the predominant factors that can influence the performance and the mechanical, sensory, biocompatibility, and drug delivery properties of HGs [28].
HGs can be manufactured with natural (e.g., alginate, chitosan, or collagen) [29], or synthetic (e.g., polyethylene glycol, polyvinyl alcohol, or polyacrylic acid) materials [30]. Cationic guar gum (CGG), a derivative of guar gum, is a polysaccharide widely used for the formation of HGs due to its low cost and easy availability [31]. Structurally, CGG is constituted by a backbone of mannose units linked by β-1,4 glycosidic bonds with galactose units attached through α-1,5 glycosidic bonds. In contrast to its guar gum counterpart, CGG is characterized by its positive nature, which arises from the presence of quaternary ammonium groups and strongly contributes to its interaction with negatively charged molecules, such as mucin [32], and influences its capability to disrupt components from the cell membranes of pathogenic microorganisms, and wound healing activity [33]. In contrast to other cationic polymers, such as alginate and dextran, CGG is preferred due to its low toxicity, biodegradability, and controlled release properties. In addition, it is advantageous due to its versatility and modifiability for chemical modification and functionalization.
The complete formation of HGs for oral mucosa delivery requires the use of crosslinking agents such as polyethylene glycol (PEG). Contrarily to other hydrophilic polymers, PEG and its branched derivatives are synthetic polymers that possess significant biocompatibility, solubility, and hydrophilicity for materials design with applications in the clinical pipeline [34]. In addition, it is a suitable raw material that is characterized by its non-immunogenicity, versatility to be modified with distinct functional groups, and stability for long-term storage and use. For the formation of HGs, PEG is crosslinked with chemical or physical agents to aid the formation of the three-dimensional network of HGs. Propylene glycol (PG) is another polymer frequently used in combination with PEG, as it can drive ionic crosslinking reactions and result in stable HG networks. For the synthesis of drug delivery systems, the use of PG is convenient as it exhibits a high dissolving capacity of organic and inorganic analytes, prevents moisture, and executes non-toxic effects.
However, additional weak organic acids such as citric acid (CA) can assist in such a reaction by creating ester linkages between PEG and PG and, hence, contribute to the stabilization of the HG structure and its mechanical features.
Toxicological analyses are required for identifying and characterizing the potential toxicity of substances and assessing dose-response relationships prior to their consideration in future pharmaceutical research. For nanobiotechnology purposes, in vitro and in vivo toxicological assays have been necessary to determine safe exposure limits and develop strategies for the safe therapeutic evaluation of silver, titanium, and zinc oxide nanoparticles [35,36,37]. In contrast to other in vivo models (e.g., Zebrafish and Drosophila melanogaster), Artemia salina, commonly known as brine shrimp, represents a major model to perform toxicity assays due to their ease of culture, sensitivity, and short life cycle [38]. In addition, it is preferred to evaluate the possible toxicity of bioactive materials such as drug delivery systems since it enables their large-scale screening through cost-effective, standardized, and simple experimental conditions.
The current study aims to optimize, develop, characterize, and evaluate HGs based on CGG/PEG for potential applications in oral mucosa. A 2k factorial experimental design was implemented to optimize the viscosity, pH, and sensory properties of HGs. The chemical composition, mass loss, and surface features of HGs were investigated by a series of analytical techniques such as Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, thermogravimetric analysis (TGA), and scanning electron microscopy (SEM). The biological activities of HGs consisted of the in vitro evaluation of their mucoadhesive properties and antibacterial activity against Gram-positive (S. aureus) and Gram-negative (E. coli and K. pneumoniae) bacteria. The potential toxicity of HGs was investigated against A. salina nauplii as an in vivo model. Results indicated that the concentration of PEG influences the sensory features, structural arrangement, mass loss patterns, mucoadhesive capability, and antibacterial activity of the developed HGs. However, it does not influence their toxicity against A. salina nauplii. The results retrieved from this work provide novel insights into CGG/PEG-based HGs with desirable optimized features, mucoadhesive and antibacterial activities, and biocompatibility for oral mucosa drug delivery.
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Materials
CGG (N-Hance™ CG13, guar hydroxypropyltrimonium chloride, medium charge density, medium nitrogen content, and high molecular weight) was donated by Ashland®. PEG-75 lanolin (Solulan™ 75 lanolin) was obtained from Lubrizol™ (Ciudad de Mexico, Mexico). CA and PG-USP were from Reactivos Meyer® (Ciudad de Mexico, Mexico) and AzuMex® (Heroica Puebla de Zaragosa, Mexico), respectively. Porcine stomach mucin-type II was obtained from Sigma-Aldrich® (St. Louis, MO, USA). All systems were prepared using water from a Milli-Q® (MQ) filtration system (Millipore, Billerica, MA, USA).
Pardo-Rendón, A.G.; Mejía-Méndez, J.L.; López-Mena, E.R.; Bernal-Chávez, S.A. Development and Evaluation of the Biological Activities of a Plain Mucoadhesive Hydrogel as a Potential Vehicle for Oral Mucosal Drug Delivery. Gels 2024, 10, 574. https://doi.org/10.3390/gels10090574
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