GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses
Abstract
1. Introduction
2. Results and Discussion
2.1. Preparation and Characterization
2.1.1. FT-IR Analysis
2.1.2. UV–Vis Analysis
2.1.3. Particle Sizes and Surface Charges
2.1.4. X-Ray Diffraction Analysis
2.1.5. Surface Morphological Analysis
2.1.6. Electrochemical Characterization
2.2. DNA-Binding Studies
2.2.1. Electrochemical Titration
2.2.2. Analysis of Thermal Denaturation
2.2.3. UV−Vis Absorption Spectral
2.2.4. Analysis of Kb and ΔG
2.2.5. Stopped-Flow Kinetics Investigation
2.2.6. Analysis of the Fast Phase
2.2.7. Analysis of the Slow Phase
2.2.8. Analysis of the Overall Reaction
2.2.9. Analysis of Drug–DNA Kinetic Stability
2.2.10. Evaluation of Nanoparticle–DNA Binding Affinity
2.3. Loading Efficiency
2.4. In Vitro Stimuli-Responsive Release
2.5. In-Vitro Drug Release Mechanism
2.6. Analysis of Kkp and n
2.7. Antibacterial Activity
2.8. Cytotoxic Activity
2.9. Analysis of Therapeutic Index
2.10. Morphological Analysis
3. Materials and Methods
3.1. Chemicals
3.2. Preparation of Chitosan Nanoparticles
3.3. Preparation of LED-Loaded Chitosan Nanoparticles
3.4. Preparation of LED-PLA-Loaded Chitosan Nanoparticles
3.5. Characterization
3.6. Procedures for DNA Binding Studies
3.6.1. Cyclic Voltammetry Measurements
3.6.2. Thermal Denaturation Measurements
3.6.3. UV–Vis Absorption Spectra
3.6.4. Stopped-Flow Experiments and Kinetic Measurements
3.7. Loading Efficiency Experiment
3.8. In Vitro Drug Release
3.9. Drug Release Mechanism
3.10. Antibacterial Assay
3.11. Cell Viability Assay
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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LED | LED@CS NPs | LED-PLA@CS NPs | |
---|---|---|---|
Mean Size | 152.4 nm | 142.5 nm | 393.7 |
PDI | 0.277 | 0.304 | 0.389 |
Zeta Potential | +9.44 mV | +43.51 mV | +33.27 |
Tm | |
---|---|
DNA free | 72.1 |
DNA-LED complex | 71.8 |
DNA-LED@CS NPs complex | 71.3 |
DNA-LED-PLA@CS NPs complex | 75.2 |
LED | LED@CS NPs | LED-PLA@CS NPs | |
---|---|---|---|
k1 [M−1 s−1] | 2.5 ± 0.3 | 5.72 ± 0.60 | 18.2 ± 2.2 |
k−1 [s−1] | 0.56 ± 0.01 | 0.22 ± 0.02 | 0.15 ± 0.07 |
Ka1 [M−1] | 4.48 | 26.0 | 120 |
Kd1 [10−2 M] | 22.0 | 3.8 | 0.83 |
ΔG1 [kJ mol−1] | −3.72 | −8.07 | −11.85 |
LED | LED@CS NPs | LED-PLA@CS NPs | |
---|---|---|---|
k2 [M−1 s−1] | 1.63 ± 0.58 | 0.24 ± 0.02 | 3.58 ± 0.3 |
k−2 [10−2 s−1] | 6.0 ± 1.0 | 0.3 ± 0.1 | 2.0 ± 0.7 |
Ka2 [M−1] | 27.2 | 80.0 | 179.0 |
Kd2 [10−2 M] | 3.67 | 1.25 | 0.55 |
ΔG2 [kJ mol−1] | −8.14 | −10.81 | −12.80 |
LED | LED@CS NPs | LED-PLA@CS NPs | |
---|---|---|---|
Kd [10−3 M] | 7.8 | 0.47 | 0.045 |
Ka [M] | 128.4 | 2131 | 22,026 |
ΔG [kJ mol−1] | −12.02 | −18.98 | −24.79 |
Kinetic Model | LED@CS NPs | LED-PLA@CS NPs | ||||
---|---|---|---|---|---|---|
pH = 7.4 | pH = 5.4 | pH = 5.4 + 10 mM GSH | pH = 7.4 | pH = 5.4 | pH = 5.4 + 10 mM GSH | |
First order | 0.989 | 0.944 | 0.935 | 0.739 | 0.784 | 0.814 |
Second order | 0.941 | 0.978 | 0.973 | 0.866 | 0.882 | 0.900 |
Higuchi | 0.977 | 0.963 | 0.943 | 0.961 | 0.968 | 0.940 |
Korsmeyer–Peppas | 0.979 | 0.987 | 0.978 | 0.992 | 0.993 | 0.981 |
LED@CS NPs | LED-PLA@CS NPs | |||
---|---|---|---|---|
n | KkP | n | KkP | |
pH = 7.4 | 0.37 | 0.14 | 0.22 | 0.24 |
pH = 5.4 | 0.42 | 0.18 | 0.23 | 0.26 |
pH = 5.4 + 10 mM GSH | 0.48 | 0.24 | 0.25 | 0.29 |
Samples | IC50 (μg ml−1) | Therapeutic Index (TI) | |
---|---|---|---|
HepG2 Cancer Cells | WI-38 Cells | ||
LED | 101.76 ± 0.33 | 184.46 ± 2.67 | 1.81 |
LED@CS NPs | 110.46 ± 1.35 | 232.85 ± 2.63 | 2.11 |
LED-PLA@CS NPs | 144.76 ± 0.69 | 341.77 ± 3.32 | 2.36 |
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Albasiony, A.M.; Beltagi, A.M.; Ibrahim, M.M.; Shaban, S.Y.; van Eldik, R. GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses. Int. J. Mol. Sci. 2025, 26, 6070. https://doi.org/10.3390/ijms26136070
Albasiony AM, Beltagi AM, Ibrahim MM, Shaban SY, van Eldik R. GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses. International Journal of Molecular Sciences. 2025; 26(13):6070. https://doi.org/10.3390/ijms26136070
Chicago/Turabian StyleAlbasiony, Ahmed M., Amr M. Beltagi, Mohamed M. Ibrahim, Shaban Y. Shaban, and Rudi van Eldik. 2025. "GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses" International Journal of Molecular Sciences 26, no. 13: 6070. https://doi.org/10.3390/ijms26136070
APA StyleAlbasiony, A. M., Beltagi, A. M., Ibrahim, M. M., Shaban, S. Y., & van Eldik, R. (2025). GSH/pH-Responsive Chitosan–PLA Hybrid Nanosystems for Targeted Ledipasvir Delivery to HepG2 Cells: Controlled Release, Improved Selectivity, DNA Interaction, Electrochemical and Stopped-Flow Kinetics Analyses. International Journal of Molecular Sciences, 26(13), 6070. https://doi.org/10.3390/ijms26136070