Cationic Polysaccharide Research Articles

Recent Advances in the Preparation, Antibacterial Mechanisms, and Applications of Chitosan

Chitosan, a cationic polysaccharide derived from the deacetylation of chitin, is widely distributed in nature. Its antibacterial activity, biocompatibility, biodegradability, and non-toxicity have given it extensive uses in medicine, food, and cosmetics. However, the significant impact of variations in the physicochemical properties of chitosan extracted from different sources on its application efficacy, as well as the considerable differences in its antimicrobial mechanisms under varying conditions, limit the full realization of its biological functions. Therefore, this paper provides a comprehensive review of the structural characteristics of chitosan, its preparation methods from different sources, its antimicrobial mechanisms, and the factors influencing its antimicrobial efficacy. Furthermore, we highlight the latest applications of chitosan and its derivatives across various fields. We found that the use of microbial extraction shows promise as a new method for producing high-quality chitosan. By analyzing the different physicochemical properties of chitosan from various sources and the application of chitosan-based materials (such as nanoparticles, films, sponges, and hydrogels) prepared using different methods in biomedicine, food, agriculture, and cosmetics, we expect these findings to provide theoretical support for the broader utilization of chitosan.

Improve anti-biofilm efficacy of ultrasound by modulating the phase transition of exopolysaccharides

This study focused on the adverse sonochemical effect of ultrasound on biofilm extracellular polysaccharide and the adaptive biofilm responses for ultrasound resistance. Results showed ultrasound triggered phase transition of polysaccharides within biofilm from solation to gelation, which induced following biofilm viscoelasticity enhancement, consequential failure of biofilm removal and bacteria killing. Introducing additional cationic polysaccharide, 1.25 % chitosan, inhibited the ultrasound responsive polysaccharides gelation and biofilm viscoelasticity enhancement, exerted synergistic antibacterial (97.40 %) and antibiofilm (96.38 %) effects with 120 W ultrasound combined on S. aureus biofilm, prolonged the preservation time of milk 2.45 times longer compared with ultrasound alone. These findings indicated the possible mechanism and solution to improve ultrasound efficacy on biofilm control and bacteria suppression, exhibit the promising prospect of ultrasound combined strategy in hygiene issues of food and medical industry

An intranasal cationic liposomal polysaccharide vaccine elicits humoral immune responses against Streptococcus pneumoniae

Diseases caused by S. pneumoniae are the leading cause of child mortality. As antibiotic resistance of S. pneumoniae is rising, vaccination remains the most recommended solution. However, the existing pneumococcal polysaccharides vaccine (Pneumovax® 23) proved only to induce T-independent immunity, and strict cold chain dependence of the protein conjugate vaccine impedes its promotion in developing countries, where infections are most problematic. Affordable and efficient vaccines against pneumococcus are therefore in high demand. Here, we present an intranasal vaccine Lipo+CPS12F&αGC, containing the capsular polysaccharides of S. pneumoniae 12F and the iNKT agonist α-galactosylceramide in cationic liposomes. In BALB/cJRj mice, the vaccine effectively activates iNKT cells and promotes B cells maturation, stimulates affinity-matured IgA and IgG production in both the respiratory tract and systemic blood, and displays sufficient protection both in vivo and in vitro. The designed vaccine is a promising, cost-effective solution against pneumococcus, which can be expanded to cover more serotypes and pathogens.

Stabilization of fermented tomato (Solanum lycopersicum L.) juice by differently charged hydrocolloids

This study investigated the impact of three different charged hydrocolloids, anionic polysaccharide (soluble soybean polysaccharide, SSPS), neutral polysaccharide (pullulan polysaccharide, PUL), and cationic polysaccharide (chitosan, CS), and their complexation on the stabilization efficiency of fermented tomato juice (FTJ). The effect of hydrocolloids on FTJ under different treatment conditions were comprehensively evaluated by determining the particle size distribution, zeta potential, rheological properties, Fourier transform infrared spectroscopy, surface tension, and LUMiSizer. The combined conditions suggest that PUL exhibits better storage stability than SSPS and CS when used individually. Compared with the use of the stabilizers, the combination of hydrocolloids had a greater impact on the storage stability of the FTJ, and the storage stability of the FTJ increased when 0.15% SSPS + 0.03% PUL + 0.15% CS was added. This study lays the groundwork for the development of stable fruit juice beverages.

Materials based on biodegradable polymers chitosan/gelatin: a review of potential applications.

Increased mass manufacturing and the pervasive use of plastics in many facets of daily life have had detrimental effects on the environment. As a result, these worries heighten the possibility of climate change due to the carbon dioxide emissions from burning conventional, non-biodegradable polymers. Accordingly, biodegradable gelatin and chitosan polymers are being created as a sustainable substitute for non-biodegradable polymeric materials in various applications. Chitosan is the only naturally occurring cationic alkaline polysaccharide, a well-known edible polymer derived from chitin. The biological activities of chitosan, such as its antioxidant, anticancer, and antimicrobial qualities, have recently piqued the interest of researchers. Similarly, gelatin is a naturally occurring polymer derived from the hydrolytic breakdown of collagen protein and offers various medicinal advantages owing to its unique amino acid composition. In this review, we present an overview of recent studies focusing on applying chitosan and gelatin polymers in various fields. These include using gelatin and chitosan as food packaging, antioxidants and antimicrobial properties, properties encapsulating biologically active substances, tissue engineering, microencapsulation technology, water treatment, and drug delivery. This review emphasizes the significance of investigating sustainable options for non-biodegradable plastics. It showcases the diverse uses of gelatin and chitosan polymers in tackling environmental issues and driving progress across different industries.

EFFECT OF CHITOSAN CONCENTRATION ON PHYSICAL CHARACTERISTICS OF EXTRACT ETHANOLIC OF BAY LEAF (Syzygium polyanthum) NANOPARTICLE PREPARED BY CROSS-LINKING METHODS

Chitosan is a natural cationic polysaccharide that could form bonds with negatively charged polyanions like sodium tripolyphosphate (STPP) as a crosslinker. One of the important factors to develop nanoparticles is the concentration of polymer. The increased polymer concentration will increase the viscosity of the solution formed, and the size of the nanoparticles created will increase. However, if the amount of polymer is too small, the particles formed are smaller, and aggregation could be formed. In this study, the ethanolic extract of bay leaf (Syzygium polyanthum) was used as a drug that has a potent anti-dyslipidemia effect by lowering cholesterol and triglyceride levels. Studies on the ethanolic extract of a bay leaf as an anti-dyslipidemia are still limited. The objective of this research was an investigate effect of the chitosan concentration used 0.6 mg/ml (F1); 1mg/ml (F2); and 1.4 mg/ml (F3) on the physical characteristics of ethanolic extract of bay leaf (Syzygium polyanthum) nanoparticles (NSPs) prepared by cross-linking methods. The result of particle size evaluation showed that the particle size was 665.1 nm ± 14.71 (F1); 180.1 nm ± 0.5; and 221.35 nm ± 1.91 (F3), while the polydispersity index F1, F2, and F3 were 0.773 ± 0.152; 0.220 ± 0.016; and 0.212 ± 0.024 respectively. The results of this study found F2 was the most optimal chitosan concentration with particle size under 200 nm, and polydispersity index under 0.5 with positive ζ-potential value. In conclusion, chitosan concentration showed has an effect on the physical characteristics of the nanoparticles.

Engineering Porosity-Tuned Chitosan Beads: Balancing Porosity, Kinetics, and Mechanical Integrity.

Chitosan, a cationic natural polysaccharide derived from the deacetylation of chitin, is known for its solubility in diluted acidic solutions, biodegradability, biocompatibility, and nontoxicity. This study introduces three innovative methods for preparing various types of porous chitosan beads: solvent extraction, surfactant extraction, and substance decomposition. These methods involve the integration and subsequent extraction or decomposition of materials during the synthesis process, eliminating the need for additional steps. We used state-of-the-art characterization techniques to analyze and evaluate the chemical and physical properties of the beads, such as Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), X-ray diffraction (XRD), and three-dimensional (3D) computed tomography (CT) scanning. The 3D CT scans visualized and measured the porosity of different bead types, ranging from 68.4% to 39.3%. This study also evaluated the mechanical properties of the particle beads under compressive forces in both wet and dry conditions, highlighting the influence of porosity on their mechanical integrity and compression pressure behavior. The adsorptive properties of these chitosan beads were studied using methylene blue as a model pollutant, emphasizing the importance of balancing porous structure, surface area, kinetics, and structural integrity. This study paves the way for the development of environmentally sustainable polymeric beads, highlighting the crucial need to balance porosity, surface area, and structural integrity to optimize their effectiveness in real-world applications.

Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

Chitosan and Chitooligosaccharides: Antifungal Potential and Structural Insights.

Chitosan is a cationic polysaccharide derived from chitin deacetylation. This polysaccharide and its oligosaccharides have many biological activities and can be used in several fields due to their favorable characteristics, such as biodegradability, biocompatibility, and nontoxicity. This review aims to explore the antifungal potential of chitosan and chitooligosaccharides along with the conditions used for the activity and mechanisms of action they use to kill fungal cells. The sources, chemical properties, and applications of chitosan and chitooligosaccharides are discussed in this review. It also addresses the threat fungi pose to human health and crop production and how these saccharides have proven to be effective against these microorganisms. The cellular processes triggered by chitosan and chitooligosaccharides in fungal cells, and prospects for their use as potential antifungal agents are also examined.

The interaction mechanism of different ionic polysaccharides with myofibrillar protein and its contribution to the heat‐induced gels

AbstractPolysaccharides are widely used as quality improvers for meat products. However, the mechanisms of how different ionic polysaccharides regulate the gelling properties of myofibrillar protein (MP) are still unclear. This study aimed to elucidate the contribution of different ionic polysaccharides to MP gelation and its mechanism. The enhancement of hydrogen bond, hydrophobic interaction, and disulfide bond between carboxymethyl cellulose sodium (CMC‐Na, anionic polysaccharide) and MP made them bind tightly, which contributes to the improvement of gel strength, water‐holding capacity, and viscoelasticity. Konjac glucomannan (neutral polysaccharide) mainly relied on physical filling to support the gel network and improve the gel characteristics. The electrostatic attraction between cationic polysaccharides and MP enhanced the binding between them. However, due to the large structure of chitosan (cationic polysaccharide) sugar chain, it can only attach to the surface of protein, which limits the interaction between them. These findings will provide guidance for the application of polysaccharides as food quality improvers or fat substitutes and the design of new low‐fat restructured meat products.

A thorough evaluation of chitosan-based packaging film and coating for food product shelf-life extension

Chitosan is a cationic polysaccharide and derived from chitin present in exoskeleton of crustaceans such as prawns, crabs, and lobsters. This natural biopolymer chemical structure is identical to cellulose but insoluble in aqueous and organic solvents. Chitosan is an ecologically beneficial biodegradable alternative to synthetic polymers, notably typical plastics. Chitosan may be used to make a variety of films, coatings, and membranes, each having its own distinct features. Chitosan films are biodegradable, translucent, and have high mechanical strength, making them adequate for food packaging, water purification, and wound dressings. Chitosan-based films and coatings are often used for packaging foods such as fruits and vegetables, meat, and seafood. According to the findings, chitosan films have outstanding barrier qualities against UV radiation, gases, and moisture. They can assist in safeguarding packed objects from outside influences such as oxidation and humidity, which could degrade their quality. Chitosan's antibacterial qualities naturally limit the development of bacteria, viruses, and fungi. Because of this property, chitosan-based films are useful for packaging perishable food products since they may extend shelf life by lowering microbial contamination. Modifying the chitosan content or adding additional components can change the mechanical strength and flexibility of the film. Surfaces may be treated with chitosan coatings to improve qualities such as water resistance, barrier properties, and antibacterial activity. This review article discusses different approaches such as solvent casting, extrusion, electrospinning, thermoplastic method, layer-by-layer assembly, spraying, dipping for the preparation of chitosan-based films and coatings. Further these food packaging material properties such as viscosity, thermal properties, optical properties, solubility, hydrophobicity, color, mechanical characteristics, barrier qualities, antibacterial properties, antioxidant activity and prolonging the shelf life of products is described in depth.

Combining different ionic polysaccharides and pH treatment improved functional properties of soybean protein amyloid fibrils through structural modifications

This paper reported the effects of different ionic polysaccharides (a cationic polysaccharide (chitosan, CTS), an anionic polysaccharide (high methoxyl pectin, HMP), and a neutral polysaccharide (dextran, DEX)) and pHs (2.0, 4.5, 7.0, and 9.0) on the structural and functional properties of soybean protein amyloid fibrils (SAF) under non-covalent interactions. The fluorescence spectroscopy, ζ-potential, surface hydrophobicity (H0), and turbidity analyses revealed that, at any pH, HMP had a greater effect on the structure of SAF than CTS or DEX. Electrostatic and hydrophobic interactions predominated between SAF and HMP. The addition of negatively charged HMP prevented the exposure of hydrophobic groups, the H0 and ζ-potential decreased. Moreover, compared to SAF, the rheological properties of SAF-HMP complexes were enhanced. In particular, at pH 2.0, HMP was crosslinked with SAF and changed the fibrils conformation, the foaming stability of SAF-HMP increased to 100%. Furthermore, at pHs 2.0 and 4.5, strong electrostatic repulsion occurred between CTS and SAF. Therefore, SAF-CTS exhibited the highest emulsifying activity index and emulsifying stability index, which are 58.21 m2/g and 41.76 min, respectively, followed by SAF-HMP. By contrast, DEX had the least influence on the fibrils. These results will give a hand for industrial production of soybean protein amyloid fibrils.

Effect of Chitosan Degradation Products, Glucosamine and Chitosan Oligosaccharide, on Osteoclastic Differentiation.

Chitosan, a natural cationic polysaccharide derived from crustaceans and shellfish shells, is known for its advantageous biological properties, including biodegradability, biocompatibility, and antibacterial activity. Chitosan and its composite materials are studied for their potential for bone tissue repair. However, the effects of chitosan degradation products, glucosamine (GlcN) and chitosan oligosaccharide (COS), on osteoclasts remain unclear. If these chitosan degradation products promote osteoclastic differentiation, careful consideration is required for the use of chitosan and related materials in bone repair applications. Here, we assessed the effects of high (500 μg/mL) and low (0.5 μg/mL) concentrations of GlcN and COS on osteoclastic differentiation in human peripheral blood mononuclear cells (PBMCs) and murine macrophage-like RAW264 cells. A tartrate-resistant acid phosphatase (TRAP) enzyme activity assay, TRAP staining, and actin staining were used to assess osteoclastic differentiation. High concentrations of GlcN and COS, but not low concentrations, suppressed macrophage colony-stimulating factor (M-CSF)- and RANKL-dependent increases in TRAP enzyme activity, TRAP-positive multinuclear osteoclast formation, and actin ring formation in PBMCs without cytotoxicity. Similar effects were observed in the RANKL-dependent osteoclastic differentiation of RAW264 cells. In conclusion, chitosan degradation products do not possess osteoclast-inducing properties, suggesting that chitosan and its composite materials can be safely used for bone tissue repair.

Exploring modified chitosan-based gene delivery technologies for therapeutic advancements

One of the critical steps in gene therapy is the successful delivery of the genes. Immunogenicity and toxicity are major issues for viral gene delivery systems. Thus, non-viral vectors are explored. A cationic polysaccharide like chitosan could be used as a nonviral gene delivery vector owing to its significant interaction with negatively charged nucleic acid and biomembrane, providing effective cellular uptake. However, the native chitosan has issues of targetability, unpacking ability, and solubility along with poor buffer capability, hence requiring modifications for effective use in gene delivery. Modified chitosan has shown that the “proton sponge effect” involved in buffering the endosomal pH results in osmotic swelling owing to the accumulation of a greater amount of proton and chloride along with water. The major challenges include limited exploration of chitosan as a gene carrier, the availability of high-purity chitosan for toxicity reduction, and its immunogenicity. The genetic drugs are in their infancy phase and require further exploration for effective delivery of nucleic acid molecules as FDA-approved marketed formulations soon.

Chitosan, chitosan derivatives, and chitosan-based nanocomposites: eco-friendly materials for advanced applications (a review).

For many years, chitosan has been widely regarded as a promising eco-friendly polymer thanks to its renewability, biocompatibility, biodegradability, non-toxicity, and ease of modification, giving it enormous potential for future development. As a cationic polysaccharide, chitosan exhibits specific physicochemical, biological, and mechanical properties that depend on factors such as its molecular weight and degree of deacetylation. Recently, there has been renewed interest surrounding chitosan derivatives and chitosan-based nanocomposites. This heightened attention is driven by the pursuit of enhancing efficiency and expanding the spectrum of chitosan applications. Chitosan's adaptability and unique properties make it a game-changer, promising significant contributions to industries ranging from healthcare to environmental remediation. This review presents an up-to-date overview of chitosan production sources and extraction methods, focusing on chitosan's physicochemical properties, including molecular weight, degree of deacetylation and solubility, as well as its antibacterial, antifungal and antioxidant activities. In addition, we highlight the advantages of chitosan derivatives and biopolymer modification methods, with recent advances in the preparation of chitosan-based nanocomposites. Finally, the versatile applications of chitosan, whether in its native state, derived or incorporated into nanocomposites in various fields, such as the food industry, agriculture, the cosmetics industry, the pharmaceutical industry, medicine, and wastewater treatment, were discussed.

Physically cross-linked chitosan gel with tunable mechanics and biodegradability for tissue engineering scaffold

Chitosan, a cationic polysaccharide, exhibits promising potential for tissue engineering applications. However, the poor mechanical properties and rapid biodegradation have been the major limitations for its applications. In this work, an effective strategy was proposed to optimize the mechanical performance and degradation rate of chitosan gel scaffolds by regulating the water content. Physical chitosan hydrogel (HG, with 93.57 % water) was prepared by temperature-controlled cross-linking, followed by dehydration to obtain xerogel (XG, with 2.84 % water) and rehydration to produce wet gel (WG, with 56.06 % water). During this process, changes of water content significantly influenced the water existence state, hydrogen bonding, and the chain entanglements of chitosan in the gel network. The mechanical compression results showed that the chitosan gel scaffolds exhibited tunable compressive strength (0.3128–139 MPa) and compressive modulus (0.2408–1094 MPa). XG could support weights exceeding 65,000 times its own mass while maintaining structural stability. Furthermore, in vitro and in vivo experiments demonstrated that XG and WG exhibited better biocompatibility and resistance to biodegradation compared with HG. Overall, this work contributes to the design and optimization of chitosan scaffolds without additional chemical crosslinkers, which has potential in tissue engineering and further clinical translation.

Characteristic of the interaction mechanism between soy protein isolate and functional polysaccharide with different charge characteristics and exploration of the foaming properties

The commercial production process will partially damage the foaming properties of soy protein isolate (SPI). Adding functional polysaccharides have a positive effect on the foaming properties of SPI, but the effects are affected by several factors. For this reason, three kinds of polysaccharides with different charges, including chitosan (CS, a cationic polysaccharide), guar gum (GUG, a neutral polysaccharide) and gellan gum (GEG, an anionic polysaccharide), were complexed with SPI at different mass ratios to discuss their interaction mechanism, and the foaming properties of these complexes were preliminarily explored. After complexation, the structure of SPI was further folded and interacted with three polysaccharides mainly through non-covalent bonding. Specifically, SPI and CS interacted through weak electrostatic interaction, volume exclusion effect and hydrogen bonding at a high CS concentration. SPI and GEG interacted through strong electrostatic interaction and hydrogen bonding. However, SPI and GUG interacted only through hydrogen bonding. In addition to SPI/CS10, the foamability of SPI/polysaccharide complexes was significantly lower than single SPI. By contrast, the foam stability of all the complexes was much higher than single SPI, and increased with the increase of the polysaccharide concentration, among which the SPI/GEG complexes showed the highest foam stability. These findings provided the interaction mechanism between SPI and polysaccharides with different charges, and the complexes with outstanding foam stability can be used for improving the stability and texture of aerated foods.

Applications of Chitosan Nanoparticles in Dentistry: A Review.

Recently, nanotechnology has garnered significant interest across various fields due to its emerging and diverse applications. Numerous investigators have proposed that chitosan nanoparticles (CSNPs) stand out as some of the most promising nanomaterials for facilitating various activities. Chitosan, a natural biopolymer established through the deacetylation of chitin, has been extensively studied using interdisciplinary approaches for a wide range of applications. Chitosan biomaterials exhibit distinctive attributes, including biodegradability, muco-adhesion, and biocompatibility, as well as a broad spectrum of antibacterial and antifungal properties. Furthermore, chitosan stands as the sole naturally occurring cationic polysaccharide, and it can be chemically tailored into various derivatives, depending on the intended role and utilization. The potential applications of chitosan are vast and intriguing, with many yet to be fully explored and understood. The unique characteristics of chitosan have sparked growing attentiveness in pharmaceutical industries and biomedical areas around the globe. The characteristics of chitosan like its biocompatibility, and anti-inflammatory effects hold the potential to yield promising outcomes in wound healing and periodontal care following tooth extractions. The objective of this study is to provide an overview of potential applications of chitosan in the field of dentistry.

Formation of Polyion Complex Aggregate Formed from a Cationic Block Copolymer and Anionic Polysaccharide.

Block copolymers (PmMn; P20M101 and P100M98) comprising poly(2-(methacryloyloxy)ethylphosphorylcholine) (PMPC, P) containing biocompatible phosphorylcholin pendants and cationic poly((3-acryloylaminopropyl) trimethylammonium chloride) (PMAPTAC, M) were synthesized via a controlled radical polymerization method. The degrees of polymerization of the PMPC and PMAPTAC segments are denoted by subscripts (PmMn). The mixture of cationic PmMn and anionic sodium chondroitin sulfate C (CS) with the pendant anionic carboxylate and sulfonate groups formed polyion complex (PIC) aggregates in phosphate-buffered saline. A charge-neutralized mixture of P20M101 with CS formed P20M101/CS PIC vesicles with a hydrodynamic radius (Rh) of 97.2 nm, zeta potential of ca. 0 mV, and aggregation number (Nagg) of 23,044. PMPC shells covered the surface of the PIC vesicles. The mixture of P100M98 and CS formed PIC spherical micelles with the PIC core and hydrophilic PMPC shells. The Rh, zeta potential, and Nagg of the PIC micelles were 26.4 nm, ca. 0 mV, and 404, respectively. At pH < 4, the carboxylate anions in CS were protonated. Thus, the charge balance in the PIC micelles shifted to decrease the core density owing to the electrostatic repulsions of the excess cations in the core. The PIC micelles dissociated at a NaCl concentration ≥0.6 M owing to the charge screening effect. The positively charged PIC micelles with excess P100M98 can encapsulate anionic dyes owing to electrostatic interaction.

Synthesis and Characterization of Polymer-Based Membranes for Methotrexate Drug Delivery.

Methotrexate or amethopterin or 4-amino-N10-methyl pteroylglutamic acid is used for treating autoimmune diseases, as well as certain malignancies. Drug delivery systems, which are based on biopolymers, can be developed to improve the therapeutic and pharmacological properties of topically administered drugs. Biopolymers improve the therapeutic effect of drugs, mainly by improving their biodistribution and modulating drug release. This study presents the synthesis of membranes based on anionic polysaccharides and cationic polysaccharides for transdermal delivery of the active ingredient methotrexate, as well as a compatibility study between methotrexate and each of the components used in the prepared membranes. The obtained membranes based on different marine polysaccharides, namely κ-carrageenan and chitosan, for the release of the active ingredient methotrexate were characterized using techniques such as TG, FTIR, UV-Vis spectrophotometry, FTIR microscopy, water absorption capacity, water vapor permeability, and biodegradation rate. Following the studies, the membranes suitable for the transdermal release of the active substance were validated.