Green Fabrication Approach to Hydrogel Synthesis by Using Natural Polymers, their Characterization, and Applications

Hydrogels are hydrophilic, three-dimensional polymeric matrixes that absorb water and resemble biological tissues. Researchers choose them for specific projects, with synthetic hydrogels offering longer service life and improved strength, while natural hydrogels are preferred for biocompatibility and biodegradability. Conventional hydrogels have low mechanical strength.¹ Polymers, made from organic sources, are versatile building blocks used in medical devices, pharmaceuticals, culinary, and cosmetic industries due to their affordability and potential biodegradability.² A polymer hydrogel is a network of hydrophilic polymers that can expand in water or biological fluids, holding significant amounts up to thousands, 20%, or 400 times their original weight.³ Hydrogels can be synthesized using various natural polymers. Hydrogels can be developed through physical, chemical, or irradiation methods, with quality influenced by factors like topologies, cross-linking patterns, end densities, polymer manufacturing method, pH, temperature, and ionic strength. Some commonly used natural polymers in hydrogel synthesis include alginate, chitosan, collagen, inulin, pectin, lignin, etc. The characterization of hydrogels involves a combination of these techniques to elucidate their physical, chemical, mechanical, and biological properties, which are critical for optimizing their performance in various applications. These natural polymers offer advantages such as biocompatibility, biodegradability, and tunable properties, making them suitable for a wide range of applications in areas such as drug delivery, tissue engineering, wound healing, and regenerative medicine.⁴

Collagen, a natural polymer, is used in hydrogels for articular cartilage strength and structure. However, its low adsorption capacity has disadvantages. Collagens are essential for tissue scaffolding, cell adhesion, malignancy, angiogenesis, and morphogenesis. Collagen bioink, a type of collagen, could revolutionize 3D printing for animal body support.⁵

Collagens are three polypeptide chains with a broadened polyproline-II shape, a straight-handed supercoil, and a residue stumble between adjacent chains. Polypeptide chains consist of a repeating triplet Gly-XY, containing glycyl residues and proline or 4-hydroxyproline, held together by interchain hydrogen bonding and encircled by organized hydration networks as shown in Figure 1. ⁶

Figure 1:
Structure of Collagen.

Collagen, derived from natural sources like animal tissues or recombinant protein synthesis, can be synthesized. It’s primarily found in Achilles tendon, bone, and skin, with skin being the sole source for research purposes.⁷ Collagen extraction removes non-collagenous material, while skin rejuvenation involves pretreatment stages like cold water bathing, skin slicing, and periodic water changes.⁸

Superabsorbent hydrogels, made from sodium polyacrylates, are water-soluble polymers used in biotechnology and pharmaceutical industries. Pectin, a naturally occurring polysaccharide found in fruits, is considered an environmentally safe alternative to synthetic polymers. Its carboxylic group makes it suitable for drug delivery and can reduce cholesterol and serum glucose levels.⁹

Pectin, a complex compound of 17 monosaccharides with 20 linkages, is found in dicotyledons’ primary cell wall and central lamella, as displayed in Figure 2. Its structure diversity is influenced by its basic structure, with the Degree of Methylation tool used.¹⁰

Figure 2:
Structural representation of Pectin.

Pectin, a plant-based polysaccharide found in fruits like sunflower heads, sugar-beet, and mango waste, serves various food industry purposes and is suitable for drug delivery.¹⁰ Thermal hydrolysis of citrus peels, lemons, limes, apple pomace, and sugar-beet plum is used for pectin extraction, but high waste and energy consumption demand green technology. ¹¹

Alginate, a long-chain hydrophilic polysaccharide derived from seaweed, was first used in cuisine around 600 B.C. and refined in 1896 by Krefting. It is used in food, medicine delivery, tissue engineering, wound dressings, and more.

Alginate is a monomeric compound consisting of two units, β-(1,4)-linked d-mannuronic acid (ManAp) and α-(1,4)-linked l-guluronic acid (GulAp), with free hydroxyl and carboxyl groups forming hydrogen bonds. The structure is shown in Figure 3.¹²

Figure 3:
Chemical structure of alginate.

Brown marine algae, treated with alkali solutions, produce alginate, a commercially available product. It is derived from various species and bacteria, including Pseudomonas and Azotobacter.¹³ Alginate extraction involves proton exchange, solubilization with alkali, filtering, precipitation, drying, and milling, leaving certain cytotoxic impurities unsuitable for biomedical applications. ¹⁴

Inulin, a naturally occurring fructan polysaccharide from Asteraceae plants, mimics fats, acts as a low-calorie sweetener, and alters texture. It’s used in pharmaceutical and food industries due to its flexible backbone and stability in the gastrointestinal tract. Inulin’s solubility changes with chain length, making it suitable for colon targeting.¹⁵

The structure as shown in Figure 4 shows that primary constituents are (2-1) fructofuranosyl units and a terminal glycopyranose unit (1-2), making it a member of the fructans family. Insulin properties are influenced by polymerization and branching. High-performance inulin, with a DP greater than 23, forms a gel structure, alters texture, and mimics fat. Branched molecules and lower DP inulin improve flavor and are used as sucrose replacements in foods.

Figure 4:
Inulin structure.

Fructans are found in 15% of flowering plant species, while inulin is derived from fruits, vegetables, cereals, roots, rhizomes, and bacteria, primarily found in Jerusalem and Chicory. Inulin can be obtained from various plants like dandelion roots, chicory roots, burdock roots, and dahlia bulbs. Inulin is produced through extraction and purification of chicory roots, followed by liming, carbonation, filtering, demineralization, sterilization, filtration, evaporated, and drying processes.¹⁶

Lignin, the second most abundant polymer, accounts for 10-30% of organic carbon in the biosphere. Found in vascular plants and angiosperms, it is a carbon and methoxyl-rich material.¹⁷ Lignocellulosic biomass, a renewable carbon source, offers sustainable biorefinery options due to its role in plant growth, nutrient transportation, and mechanical support.

Lignin, a complex aromatic heteropolymer, consists of three monomers: p-coumaryl alcohol, guaiacyl aclochol, and sinapyl alcohol, with hardwood lignin having a lower molecular weight as shown in Figure 5. Its functional groups form linkages during biosynthesis.¹⁸

Figure 5:
The three monolignols considered as building block of lignin.

This led to the increase in plant size, resulting in their dominance in the terrestrial environment Lignin is a bioproduct used in pretreatment and separation processes for lignocellulosic biomasses, including pulp and second-generation ethanol production, generated by agro-industrial activities.¹⁹ The LignoBoost process is an efficient and cost-effective method for lignin extraction from black liquor, involving CO₂ injection, precipitation, filtering, acid filtration, crushing, and drying to form lignin powder, with higher pH decreases increasing precipitated yield but increasing CO₂ gas consumption and operational costs.²⁰

Chitosan, a natural linear bio-polyaminosaccharide found in crustaceans’ exoskeletons and shellfish shells, is used as a fat magnet, cheap, biodegradable, non-toxic, and ideal for hydrogel structures due to its hydrophilic nature, biodegradability, and biocompatibility.²¹

Chitosan is a random copolymer with a molar fraction of beta-(1à4)-N-acetyl-D-glucosamine and a fraction (1-DA) of beta-(1à4)-D-glucosamine, characterized by its degree of acetylation.²² Chitin, unlike cellulose, shares an acetamide group at the carbonyl group, making it nearly identical to cellulose due to its chemical backbone. The structure is shown in Figure 6.

Figure 6:
Chemical structure of chitin and chitosan.

Chitin, a chitosan compound, is found in crustaceans, fungi, insects, and cell walls, with 15-30% content in crab cuticles, exoskeletons, and fungi cell walls.²³ Chitosan is a natural product of decomposing chitin in shells, obtained by collecting, demineralizing, washing, and de-proteinizing prawn shell waste. It forms a chitin cake when excess water is removed, then treated with NaOH.²⁴

Researchers are developing hydrogels with high water absorption rates and enhanced wet strength for various applications, including drug entrapment, personal hygiene, agriculture, construction, food packaging, electronics, and cosmetics. Tragacanth, a water-soluble Asian gum component, is used in OH pharmaceuticals, cosmetics, adhesives, and more. Graining gum tragacanth with vinyl monomers produces diverse polymers with varying properties and applications.²⁵

GT, a thick gel made of soluble tragacanthin and insoluble bassorin as shown in Figure 7, is diverse in quality and functional features due to the ratios of TGSW and BIW.

Figure 7:
The precise and accurate nomenclature of the GT fraction.

Tragacanth gum, a high-molecular-weight polysaccharide from Astragalus gummifer stems, is a popular natural emulsifier and thickener in food, pharmaceuticals, cosmetics, textiles, and food thickening due to its effective emulsifying abilities, long shelf life, heat stability, and pH range, as well as its water-insoluble, gel-like component, bassorin.²⁶

Tragacanthin, a gel made of tragacanthic acid, arabinogalactan, and sugars, dissolves in water and forms a thick hydrosol. It contains varying amounts of fucose, glucose, galacturonic acid, galactose, xylose, arabinose, and rhamnose, with varying ratios in different species.²⁷ Astragalus tree wound sap is collected using tap roots with central gum cylinders, with poor-quality gum often produced due to dry conditions. The gummosis process releases soft gum and dries on the trunk using vertical, horizontal, and diagonal incision methods. Raw gum is sorted into color, ribbon, or flake morphologies.²⁸

The hydrolyzed collagen was dissolving, and the insoluble phosphate salt was removed. The mixture was stirred in a three-neck reactor at 80°C, supplemented with initiator solution, and then added with a weight ratio of AAM/AA and MBA.

After 60 min, 1 N NaOH solution was added, followed by an additional non-solvent ethanol. The mixture was stirred for 2 min to dewater, and the hydrogel was cut into small pieces. After 24 hr, the hydrogel was kept in new ethanol. The hydrogel was then baked for 10 hr at 60°C to dry.²⁹

Pectin with varying esterification degrees was used for free radical polymerization to developed an adsorbent hydrogel, which was analyzed using FTIR, SEM, and XPS. The mixture of water and pectin was mixed, homogenized with glutaraldehyde and HCl, heated at 50°C for 24 hr, then diluted with NaOH and methanol. The precipitate was filtered, dried, and finely chopped to produce off-white granules, which were then screened through a 16-mesh sieve.³⁰

Ionic crosslinking is the most effective method for creating alginate hydrogels, which can be achieved through chemical and physical crosslinking procedures. Alginate gelation and crosslinking involve the exchange of sodium ions from glucuronic acid units with divalent cations like calcium, strontium, and barium. These hydrogels have varying degrees of stability, permeability, and strength, and their mechanical properties can limit their application for long-term cell investigations. Stability depends on the exchange between monovalent cations from the environment and divalent cations from the hydrogel and is affected by alginate characteristics, concentration, source, crosslinking intensity, and molecular weight. In vitro stability varies, with calcium-crosslinked hydrogels losing stability in solutions containing chelators. However, in cell culture media, calcium alginate hydrogels can remain stable for weeks.³¹

The inulin hydrogel was developed by repeating units of monomer in the Inulin-Divinylsulfone Derivative (INUDV). The INUDV derivative was dissolved in Phosphate-Buffered Saline (PBS) and degassed under vacuum. The mixture was mixed and kept for 4 hr for crosslinking. After treating with acetone, a fine powder was produced, which was dried in P₂O₅ to achieve a consistent weight. The microparticles were mixed with a concentrated ethanolic solution of flutamide and stirred for three days. The sample was then washed with acetone to remove externalized flutamide.³²

Lignin can be employed as biomass when it is immobilised inside a polymer matrix, and a physical combination is simply lignin dispersed or blended with one or more other polymers. This strategy aims to enhance the mechanical characteristics of already prepared polymeric networks by taking use of lignin’s fundamental function in nature, which is to provide plants with structural strength. Nevertheless, very little research has been done on the semi-Interpenetrating Polymeric Networks (IPNs) that are so produced. For instance, in the presence of lignin, Peñaranda et al. used a free radical reaction to polymerize and crosslink Acrylamide (AAm) with the crosslinking agent N,N′-methylene-bis-acrylamide (NMBA) (other combinations with starch and peat were also analysed).³³

One approach that is seen to be promising is the development of hydrogel using components from natural products. Using this method, Yu and colleagues produced a hydrogel for food packaging applications based on chitosan. After being well stirred to dissolve the different weight percentages of chitosan in acetic acid, the mixture was moved to a Petri dish and kept at 70°C. After being removed, the clear layer that resembled a thin film was employed for more research (Figure 1). The hydrogel film produced by this process has a thin sheet with excellent toughness and antiadhesion properties. It is based on polysaccharides and has exceptional mechanical and antiadhesion properties.³⁴

Gum tragacanth’s active-OH group-containing arabinose units undergo graft copolymerization with acrylic acid, starting with lipase. A homogenous mixture is developed, adjusted for water absorption, and the hydrogel is extracted using water. The hydrogel is dried at 55°C.³⁵

Hydrogels’ water holding capacity is primarily due to hydrophilic groups in polymer chains, varying from 10% to thousands of times its weight. The % age swelling, a measure of water imbibed within a hydrogel, is crucial for biomedical applications, as it directly influences solute diffusion properties. Experimentally, it can be determined using the weight difference method.

where Ws is the ( weight of swollen gel) and Wd is (the weight of dry gel).³⁶

The hydrogel film is sterilized, cut into 22 cm squares, and placed on cultured tryptose soy agar. Bacteria are sprayed, and the films are incubated at 30°C for two weeks. The hydrogel film is sterilized, cut into 22 cm squares, and placed on cultured tryptose soy agar. Bacteria are sprayed, and the films are incubated at 30°C for two weeks.³⁷

A biocompatibility test is crucial for medical technology and products to ensure user safety and patient safety. Cytotoxicity assessment is essential for hydrogels used in wound dressing applications. The test involves placing the substance near host cells and incubating at 37°C or immersing it in a physiological solution. Hydrogels are sterilized, seeded with host cells, and incubated for 1 hr to promote cell growth. Microscopy or the MTT assay can be used to observe cell growth and proliferation.³⁸

Hydrogels’ network architecture allows for nutrient, compound, and cell-signaling diffusion while retaining cells as a scaffold. Crosslinking density influences diffusion properties. Evaluation methods like differential scanning calorimetry and mid-IR spectroscopy are challenging due to high water content.³⁹

Hydrogels facilitate diffusion of nutrients, compounds, and cell signals, retaining cells as a scaffold. Evaluation methods like differential scanning calorimetry and mid-IR spectroscopy are challenging due to high water content.

The study uses FTIR analysis to characterize hydrogels and hybrid networks, demonstrating the effectiveness of a proposed method for creating nanostructured materials. The method uses diffuse reflectance spectroscopy (DRIFTS-FTIR) and ATR crystals, with 32 scans obtained at 2 cmK1 resolution.⁴⁰

X-ray Diffraction (XRD) is a technique used to identify a substance’s crystal structure, revealing properties like crystallite dimension, orientation, and stresses. It uses monochromatic X-rays to diffract rays, calculates interplanar atomic spacing, and can characterize materials quantitatively and qualitatively. XRD can also describe semi-crystalline polymers with both amorphous and crystalline components.⁴¹

Scanning Electron Microscopy (SEM) is a technique used to analyze the morphological properties of hydrogels, enabling the detection of their microscale porosity with varying diameters. Low vacuum mode of a high-resolution scanning electron microscope allows for the identification of hydrogel surface morphology and monitoring of biological material surface topography without sputter-coating.⁴²

The study examines the biodegradability of hydrogel materials, specifically nanoemulsion-functionalized hydrogels, to determine their impact on enzyme activity, which is tested by mixing the hydrogel with a dissolved enzyme in PBS.⁴³

Scientists are using rheological attributes to characterize food-related products like yogurt as hydrogels, based on the correlation between molecular weight and zero shear viscosity in the food sector. This technique involves testing dynamic viscoelasticity in a humidity chamber at 37°C to minimize water evaporation.⁴⁴

Thermal analysis, thermogravimetry, and differential scanning spectroscopy are essential tools in the pharmaceutical sector for characterizing polymeric materials, understanding properties like stability and polymorphism, and ensuring product quality control. These methods reveal degradation events, particularly when produced from multiple monomers.⁴⁵

Hydrogels are effective drug delivery systems for administering immunoglobulins and proteins, with in vitro studies frequently conducted to determine their release. The medium was diluted to 900 mL, stored at 37±0.5°C, and the desired dosage form was packed in muslin cloth and placed in a dissolution basket. A 5cm diameter, 4 cm height cylinder was used as a sample holder to place the desired dosage form, preventing it from sticking to the vessel’s bottom.⁴⁶ Samples were replaced with fresh medium every hour to maintain sink conditions and maintain temperature at 37°C. The desired drug concentration was measured using a UV-vis spectrophotometer.⁴⁷ Calibration curves of desired drug was obtained for estimation of amount of released drug.

In vivo drug release investigations use HPLC and radioactivity measurement to determine residual drug contents. In vivo imaging techniques track fluorescently labeled materials’ behavior, improving data quality and reducing animal requirements. A study using mice anesthetized with ketomin and xylazine found that drug release from hydrogel disks occurs due to reduced fluorescence intensity at the implantation site. Unloaded hydrogel drugs also produce fluorescence signals, indicating sustained drug entrapment in polymeric networks. The study highlights the importance of in vivo imaging techniques in determining drug contents and improving data quality.⁴⁸

Hydrogel discs dissolve in water due to osmotic pressure gradient, swelling, and drug release. The most effective model for best release data was found to be close to 1.

The study examined drug release kinetics using various models including the 1^st order model, zero order model, Higuchi model, Korsemeyer Peppas model, and Hamilton-Crowell model.⁴⁹ The Weibull model, Baker Lonsdale model, Hopfenberg model, and Gompertz model are all statistical models used to analyze data.⁵⁰

In the field of treating skin injuries, collagen-based hydrogel is frequently employed.⁵¹ Collagen-based hydrogel films, infused with metal nanoparticles like Ag and Cu, are utilized as drug delivery devices, exhibiting antibacterial properties against common bacteria.⁵² The ubiquitous use of the designed collagen-based substitutes is because of the extensive distribution of collagen within all organs and tissues.⁵³ Collagen and doxycycline, combined for tissue recovery and bacteriostatic effects against bacteria, are expected to create a more effective local drug delivery system for treating skin infections and tooth decay.⁵⁴

Hydrogels have various applications in drug delivery, peptides, proteins, hormones, agriculture, horticulture, biotechnology, cell construction, pharmaceuticals, and biomedical fields.⁵⁵ Composite hydrogels, made from polysaccharide biopolymers and natural pectin, enhance hemostasis properties, antibacterial activity, and degradability, while maintaining anti-inflammatory and antioxidant benefits in medicine.⁵⁶ Pectin, a structural polysaccharide derived from plant cell walls, is being utilized for skin repair and regeneration due to its water solubility, biocompatibility, biodegradability, and anionic nature. Its branching polymer offers multiple target areas for chemical modification and can degrade both in vitro and in vivo.

The success of a therapeutic factor encapsulation system is significantly influenced by the coating material, with alginate being a promising option due to its non-toxic, biocompatible, and biodegradable properties.⁵⁷ Oxidized alginate-based hydrogels, being biodegradable materials, are widely utilized in tissue engineering due to their faster degradation rate compared to native alginate.⁵⁸ The preparation of thin hydrogel films made of alginate and aloe vera gel demonstrated enhanced absorption and swelling properties.⁵⁹ Alginate and carrageenan, in various forms like beads and fibers, have shown significant potential as cell-carrier materials for tissue engineering and regenerative medicine applications.⁶⁰

Inulin, a biodegradable polysaccharide, is widely used in food and pharmaceutical industries due to its biocompatibility, non-toxicity, and adaptability, making it versatile for medical applications. Longer chain inulin is suitable for drug delivery systems due to its non-solubility at normal body temperature, while shorter chain inulin is soluble and suitable for renal function tests. Inulin, a hydrophobic domain in polymeric micelles, is utilized in the administration of hydrophobic drugs like curcumin. It contributes to pH-sensitive lysosome-triggered drug delivery systems, improves drug efficacy, and can enhance drug solubility in solid dispersion.⁶¹

Lignin-based hydrogels are widely used in agriculture and industry for water treatment, serving as smart biomaterials for water retention and treatment. Lignin-based hydrogels are widely used for absorption of organic and inorganic materials due to their ability to swell water, with their absorption capacity varying depending on the hydrogel’s structure. Lignin-based hydrogels are widely used for controlled release of functional materials and water, extending soil retention and absorbing significant amounts for plant growth in sandy soils. Smart materials with stimuli response capabilities, such as pH, thermos, and mechanical responses, have increased potential for controlled release of drugs and pesticides.⁶²

Chitosan-based hydrogels, like HemCon bandage, are widely used as homeostatic and wound dressing agents to promote wound healing. Chitosan-based hydrogels are extensively utilized in tissue engineering due to their significant impact on cell adhesion, proliferation rate, and swelling properties. Chitosan-based hydrogels are utilized for oral drug delivery to the stomach, intestine, and colon, specifically for treating inflammatory bowel disease and preventing irritability.⁶³

TG is ideal for encapsulation in food processing, cosmetics, and medication manufacturing due to its non-toxic and biocompatibility properties, ensuring material protection against oxygen, light, moisture, and extreme temperatures. Industrial waste production is influenced by pollution and effluent, with plant-based chemicals like GT providing high safety, low cost, and simple processing for removing dyes and heavy metals. The soil-release capacity of an interpenetrating polymer network based on GT for spreading fertilizer across the ground, finding a progressive release profile and 79% biodegradability under burial conditions. A progressive release profile and 79% biodegradability of an interpenetrating polymer network based on GT for spreading fertilizer across the ground.⁶⁴

This study delves into the utilization of natural polymers in hydrogel synthesis, preparation methods, and characterization. This study explores both physical and chemical cross-linked hydrogels, including their kinetic modeling, and investigates their diverse applications across various fields. Additionally, the research scrutinizes drug release studies, kinetic modeling, and the wide-ranging applications of hydrogels. By combining original research papers and review articles, the study aims to offer a comprehensive insight into hydrogels as a noteworthy drug delivery system.

We would like to extend our sincere gratitude to the Deanship of the Faculty of Pharmaceutical Sciences at Lahore University of Biological & Applied Sciences (UBAS) for providing the essential resources and support that made this research possible. Their commitment to fostering academic excellence has been invaluable.