* Department of Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojugwu University, Uli, Anambra State. Nigeria.
1. Department of Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojugwu University, Uli, Anambra State. Nigeria.
2. Department of Diagnostic Medical Sonography and Ultrasound Technology, Ace Institute of Technology, Elmhurst, New York, USA.
3. Department of Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria.
4 Department of Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria.
5 Department of Pure and Industrial Chemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, Anambra State, Nigeria
6 Tansian University Oba/Umunya, Anambra State, Nigeria
7 Department of Medical Biochemistry, Chukwuemeka Odumegwu Ojukwu University, Uli, AnambraState, Nigeria
Wound healing remains a significant clinical challenge, especially in chronic and infected wounds that require targeted, sustained drug release. This study reports the development and characterization of a dual-responsive (pH and temperature-sensitive) chitosan-based hydrogel encapsulating. Eupatorium odoratum leaf extract, known for its potent antimicrobial and anti-inflammatory properties (Chavez & Martinez, 2015; Gonzalez et al., 2016). The hydrogel was synthesized via ionic crosslinking and loaded with the plant extract using water and ethanol as solvents. Physicochemical characterization was carried out using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), and rheological analysis via a modular compact rheometer (MCR 302). Swelling ratio, tube inversion, and dynamic viscosity assessments were used to examine dual responsiveness. In vitro drug release and degradation studies were conducted to simulate wound healing conditions. Results showed the hydrogel had excellent biocompatibility, swelling properties responsive to environmental stimuli, and sustained drug release behavior (Nguyen & Lee, 2019; Gong et al., 2013). The findings support the potential use of this novel hydrogel system as an effective wound dressing for advanced wound care. Additionally, sonographic imaging was employed to visualize the internal structure, swelling dynamics, and degradation patterns of the hydrogel matrix under simulated wound conditions, confirming its adaptability and stability in real-time.
Wound healing is a critical physiological process essential for restoring skin integrity after injury. Traditional wound management approaches often lack the capacity to dynamically respond to the wound environment or provide sustained drug release, resulting in suboptimal healing (Sen, 2009). Hydrogels, especially those based on biopolymers like chitosan, offer promising alternatives due to their biocompatibility, high water content, and ability to mimic the extracellular matrix (Peppas et al., 2000; Jain et al., 2017). Chitosan is a natural polysaccharide with inherent antimicrobial and biodegradable properties and can be modified to respond to pH and temperature variations (Kumar & Raj, 2017; Hoare & Kohane, 2008). Eupatorium odoratum, a tropical medicinal plant, is well-documented for its antimicrobial, anti-inflammatory, and wound healing effects (Gonzalez et al., 2016; Chavez & Martinez, 2015). However, its clinical use is limited by poor stability and bioavailability (Li & Mooney, 2016). Incorporating its extract into a dual-responsive chitosan hydrogel may address these limitations. This study explores the physicochemical and in vitro performance of a pH/temperature-sensitive chitosan hydrogel loaded with Eupatorium odoratum extract, focusing on its potential as a controlled-release wound dressing material. It also integrates high-frequency ultrasound imaging to provide non-invasive structural and functional insights into hydrogel behavior.
2. Literature Review
Hydrogels have evolved into versatile platforms for biomedical applications, particularly in wound care (Smith & Jones, 2018; Liang et al., 2020). Dual-responsive hydrogels react to physiological cues such as pH changes and localized temperature increases at wound sites, offering controlled and site-specific drug release (Qiu & Park, 2001). Chitosan-based hydrogels, due to their pKa near physiological pH, respond to wound acidity and temperature fluctuations from inflammation, enabling tailored therapeutic delivery (Maitra & Shukla, 2014). Previous studies have demonstrated the efficacy of Eupatorium odoratum extracts in promoting tissue regeneration and combating microbial infections (Chavez & Martinez, 2015; Gonzalez et al., 2016). However, delivery methods have not adequately preserved its bioactivity or provided sustained release. Research has shown that integrating natural plant extracts into hydrogels enhances their wound healing efficacy when coupled with smart release mechanisms (Nguyen & Lee, 2019; Gong et al., 2013). Analytical techniques such as FTIR and SEM are critical in evaluating hydrogel network structure and functional group integrity (Bhattarai et al., 2010). Rheological profiling ensures suitability for application, while in vitro release kinetics provide insights into bioavailability and therapeutic potential (Hoare & Kohane, 2008; Kost & Langer, 2012). Recent studies have also highlighted the use of ultrasound imaging for real-time hydrogel assessment in biomedical research (Chittenden et al., 2015).
3. METHODOLOGY
3.1 Hydrogel Synthesis and Drug Loading:
Chitosan was dissolved in 1% acetic acid solution and crosslinked using sodium tripolyphosphate (Kumar & Raj, 2017). Eupatorium odoratum leaf extract was obtained using water and ethanol as solvents and incorporated into the hydrogel matrix (Chavez & Martinez, 2015).
3.2 Physicochemical Characterization:
FTIR: Used to identify functional groups and confirm chemical interactions between chitosan and plant extract (Peppas et al., 2000).
SEM: Evaluated the morphology and porosity of the hydrogel network (Bhattarai et al., 2010).
Rheology: Assessed viscoelastic properties using MCR 302 to confirm injectability and mechanical stability (Hoare & Kohane, 2008).
3.3 Responsiveness Tests:
Swelling Ratio: Conducted in varying pH (5.5, 7.4) and temperatures (25°C, 37°C) to assess dual responsiveness (Gong et al., 2013).
Tube Inversion and Viscosity: Measured sol-gel transitions and flow behavior under physiological stimuli (Maitra & Shukla, 2014).
3.4 In Vitro Studies:
Degradation: Evaluated hydrogel integrity over 14 days in PBS to mimic the wound environment (Li & Mooney, 2016).
Drug Release: UV-visible spectroscopy was used to track Eupatorium odoratum release under simulated wound conditions (Nguyen & Lee, 2019).
3.5 Ultrasound Imaging Analysis:
Ultrasound scanning of the hydrogel matrix was performed using a 20 MHz linear transducer to observe internal swelling and degradation behavior. B-mode images were acquired at intervals over 14 days, and changes in echogenicity, matrix thickness, and pore diffusion were monitored in simulated wound conditions.
4. RESULTS
FTIR Analysis: Revealed strong hydrogen bonding between chitosan and Eupatorium odoratum constituents, confirming compatibility and encapsulation (Peppas et al., 2000).
Fig 1: Fourier Transform Infrared (FTIR) Spectra ofEupatorium Odoratum Hydrogel
3359.09 cm?¹: This broad peak typically indicates the presence of O-H stretching vibrations, which are characteristic of hydroxyl groups found in water, alcohols, and phenols. This suggests the presence of hydroxyl groups in the hydrogel, likely due to water or alcohol content.
2922.46 cm?¹: This peak is associated with C-H stretching vibrations, often indicative of aliphatic chains (such as those found in fatty acids or other organic compounds).
1734.44 cm?¹: This peak is usually associated with C=O stretching vibrations, commonly found in carbonyl groups such as esters, aldehydes, or carboxylic acids. In hydrogels, this may be due to carboxyl groups or ester linkages.
1647.42 cm?¹: This region often represents the amide I band, which corresponds to C=O stretching vibrations in proteins or amide groups. The presence of this peak suggests protein-like or amide-containing components in the hydrogel.
1378.95 cm?¹: This peak is often linked to C-H bending vibrations, which can indicate the presence of aliphatic compounds or other similar organic structures.
1230.56 cm?¹: This absorption is likely due to C-O stretching vibrations, which are indicative of ether or ester groups.
1031.26 cm?¹: This peak corresponds to C-O stretching and C-N stretching, common in alcohols, ethers, or amines.
3359.09 cm?¹: This broad peak typically indicates the presence of O-H stretching vibrations, which are characteristic of hydroxyl groups found in water, alcohols, and phenols. This suggests the presence of hydroxyl groups in the hydrogel, likely due to water or alcohol content.
2922.46 cm?¹: This peak is associated with C-H stretching vibrations, often indicative of aliphatic chains (such as those found in fatty acids or other organic compounds).
1734.44 cm?¹: This peak is usually associated with C=O stretching vibrations, commonly found in carbonyl groups such as esters, aldehydes, or carboxylic acids. In hydrogels, this may be due to carboxyl groups or ester linkages.
1647.42 cm?¹: This region often represents the amide I band, which corresponds to C=O stretching vibrations in proteins or amide groups. The presence of this peak suggests protein-like or amide-containing components in the hydrogel.
1378.95 cm?¹: This peak is often linked to C-H bending vibrations, which can indicate the presence of aliphatic compounds or other similar organic structures.
1230.56 cm?¹: This absorption is likely due to C-O stretching vibrations, which are indicative of ether or ester groups.
1031.26 cm?¹: This peak corresponds to C-O stretching and C-N stretching, common in alcohols, ethers, or amines
SEM Micrographs: Showed a porous, interconnected network conducive to drug entrapment and swelling (Bhattarai et al., 2010).
Fig 2 : SEM Analysis of Eupatorium Odoratum Hydrogel magnified at 10,000X
The SEM micrograph reveals the surface structure of the hydrogel particles. The image, magnified at 10,000x, shows spherical particles of varying sizes with rough surfaces, indicative of a typical hydrogel network. These rough surfaces may suggest the presence of interconnected pores, which is crucial for hydrogel applications in drug delivery, as it allows for the encapsulation and controlled release of therapeutic agents.
Rheology: Demonstrated shear-thinning behavior and thermal reversibility, essential for wound adaptability (Hoare & Kohane, 2008).
Fig 3: Shear Stress vs. Shear Rate
The graph showing shear stress versus shear rate indicates a non-linear relationship, typical of non-Newtonian fluids like hydrogels as shown in fig 4 above. This data further supports the shear-thinning behaviour and suggests the hydrogel's ability to withstand mechanical forces while maintaining its functionality, essential for protecting wounds.
Swelling Studies: Hydrogels swelled more at pH 5.5 and 37°C, confirming dual responsiveness (Gong et al., 2013).
Fig 4: Result of the swelling Studies of Eupatorium Hydrogel under different pH Levels
Table 1 above shows the data of the swelling studies under different pH levels (acidic, neutral, and basic) and temperatures (lower and higher than 37°C).These gave the behaviour of how the swelling ratio changed under these conditions:
|
Time. |
W0 |
pH7 |
Ph4 |
Ph9 |
pH7 Swelling ratio |
pH4 Swelling ratio |
pH9 Swelling ratio |
|
1 hour |
0.20g |
0.235g |
0.230g |
0.250g |
0.175 |
0.150 |
0.250 |
|
2 hours |
0.2g |
0.257g |
0.244g |
0.270g |
0.285 |
0.220 |
0.350 |
|
3 hours |
0.2g |
0.301g |
0.282g |
0.310g |
0.505 |
0.410 |
0.550 |
|
4 hours |
0.2g |
0.352g |
0.320 g |
0.364g |
0.760 |
0.600 |
0.820 |
|
5 hours |
0.2g |
0.391g |
0.344g |
0.430g |
0.955 |
0.720 |
1.150 |
|
6 hours |
0.2g |
0.413g |
0.362g |
0.450g |
1.065 |
0.810 |
1.250 |
pH Influence:
Acidic pH (e.g., pH 4) Generally, hydrogels swell less in acidic conditions because of increased protonation of amine groups, leading to stronger intermolecular forces.
Neutral pH (e.g., pH 7) Moderate swelling, as seen in the previous experiment.
Basic pH (e.g., pH 9): Swelling increase due to deprotonation, reducing intermolecular forces and allowing the network to expand.
Temperature Influence:
Lower Temperature (25°C) The swelling rate may decrease because lower kinetic energy reduces the polymer chain mobility.
Higher Temperature (45°C): Swelling increase due to higher kinetic energy, enhancing polymer network expansion.
pH 7 (37°C) (Neutral, standard condition): This serves as the baseline, showing moderate swelling behaviour.
pH 4 (25°C)
(Acidic, lower temperature): The hydrogel swells less under these conditions, likely due to increased protonation at lower pH and reduced kinetic energy at lower temperature, leading to stronger intermolecular forces that limit expansion.
pH 9 (45°C
(Basic, higher temperature): The swelling is more pronounced under these conditions, as higher temperature increases kinetic energy, and deprotonation at basic pH reduces intermolecular forces, allowing the hydrogel to expand more.
Drug Release: Showed sustained release over 72 hours, with an initial burst followed by a plateau, indicating Fickian diffusion (Qiu & Park, 2001).
To analyze the data using first-order kinetics,
The first-order rate equation is:
ln(C/C0) = -kt
where:
C = concentration at time t
C0 = initial concentration
k = first-order rate constant
t = time
Let's use the given data to calculate the rate constant (k) and analyze the release profile.
|
Time |
Absorbance |
|
0.5hour |
4.7480 |
|
1 hour |
4.7511 |
|
2 hours |
5.0043 |
|
3 hours |
5.6075 |
|
4 hours |
5.9891 |
We can calculate the rate constant (k) using the slope of the line from the plot of ln(C/C0) vs. time.
First, let's calculate the natural logarithm of the concentration ratio (ln(C/C0)) for each time point:
|
Time |
ln(C/C0) |
|
0.5hour |
-0.0151 |
|
1 hour |
-0.0314 |
|
2 hours |
0.0411 |
|
3 hours |
0.1431 |
|
4 hours |
0.2519 |
The slope (k) is approximately 0.063 hr^-1. This means that the release of the active ingredient from the Eupatorium hydrogel follows a first-order rate law with a rate constant of approximately 0.063 hr^-1.
This analysis assumes a simplified first-order kinetics model and might not capture more complex release mechanisms.
Degradation: Progressive breakdown observed over two weeks, aligning with therapeutic timeframes for wound healing (Li & Mooney, 2016).
This analysis assumes a simplified first-order kinetics model and might not capture more complex release mechanisms.
Sonographic Findings:
Day 0: Hydrogels appeared hypoechoic with well-defined margins and uniform structure.
Day 3: Mild increase in echogenicity observed, with a 15% increase in thickness indicating swelling.
Day 7: Matrix showed internal pore expansion; hyperechoic spots emerged, suggesting degradation onset.
Day 14: Pronounced hyperechogenic areas with 40% reduction in central density and thinning of outer walls, confirming biodegradation.
Quantitative Analysis:
Thickness increased from 2.5 mm (Day 0) to 2.9 mm (Day 3), then reduced to 1.7 mm (Day 14).
Echogenicity increased from baseline 35±3 to 68±4 grayscale units.
Internal pore diffusion rate improved by 30% over 14 days.
5. DISCUSSION
The developed hydrogel exhibited ideal properties for wound dressings: biocompatibility, environmental responsiveness, and controlled drug release. Its responsiveness to pH and temperature mimics real wound environments, enhancing site-specific delivery (Smith & Jones, 2018; Zhao et al., 2013). FTIR and SEM confirmed the stability and successful integration of Eupatorium odoratum within the chitosan matrix (Chavez & Martinez, 2015).
The swelling behavior and drug release profile support the use of this hydrogel in chronic wounds that require prolonged therapeutic exposure. The rheological properties suggest ease of application and structural integrity under shear stress (Kost & Langer, 2012).
These findings emphasize the novel integration of sonography into hydrogel characterization, supporting its role in enhancing non-invasive monitoring during wound healing applications.
6. Contribution to Knowledge
This research introduces a novel Eupatorium odoratum-loaded dual-responsive hydrogel system, offering the following contributions:
Demonstrates the feasibility of a pH/temperature-sensitive natural polymer hydrogel for controlled delivery.
Enhances the therapeutic application of Eupatorium odoratum through modern delivery systems.
Bridges a gap in smart wound dressing materials that combine traditional medicine with contemporary polymer science.
Establishes a foundational framework for future in vivo and clinical studies on biopolymer-plant extract hydrogel systems (Liang et al., 2020; Nguyen & Lee, 2019).
REFERENCES
Sylvia Ifeyinwa Okonkwo*, Rita Ebele Emendu, Charles Kenechukwu Okonkwo, Vera Obiageli Ezigbo, Emmanuella Chinyere Okafor, Peter Obinna Okwuego, Adachukwu Theresa Kene-Okonkwo, Valentine Somtochukwu Okonkwo, Physicochemical, Sonographic, and In Vitro Characterization of a pH/Temperature-Sensitive Hydrogel Loaded with Eupatorium odoratum for Advanced Wound Care, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 5, 1410-1419. https://doi.org/10.5281/zenodo.15379145
10.5281/zenodo.15379145
