Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.1.1. Chemicals
2.1.2. Immortalized Human Keratinocytes Cells (HaCat)
2.1.3. Animals
2.2. Methods
2.2.1. Preparation of NPs
2.2.2. Preparation POLX-Based Hydrogel
2.2.3. NPs Characterization
2.2.4. Surface and Morphological Analysis
2.2.5. Determination of Encapsulation Efficiency (EE)
2.2.6. Physical Stability of Insulin Nanoformulation over Time
2.2.7. Protein Structural Stability after Encapsulation
SDS-PAGE
Circular Dichroism
2.2.8. Release Behavior of Insulin Formulations over Time
In Vitro Diffusion Assay Using Franz Cells
2.2.9. Preliminary In Vitro Safety Assessment
In Vitro Assessment
2.2.10. Preliminary In Vivo Efficacy Assessment
2.2.11. Statistical Analysis
3. Results
3.1. NPs Characterization: Size, PI, Zeta Potential, Particle Morphology, and EE
3.2. Physical Stability of Nanoformulation over Time
3.3. Protein Stability after Encapsulation Process
3.4. Protein Recovery after Encapsulation Process
In Vitro Release Assay
3.5. Preliminary Safety and Efficacy Assessments
3.5.1. In Vitro Safety Assessment
3.5.2. In Vivo Preliminary Efficacy Assessment
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Formulations and Parameters | Size (nm) | PdI | Zeta Potential (mV) | EE (%) | pH |
---|---|---|---|---|---|
Empty NPs | 223 ± 1 | 0.19 ± 0.01 | −1.03 ± 0.26 | - | 5.5 ± 0.1 |
Insulin-loaded NPs | 557 ± 19 | 0.38 ± 0.02 | 0.07 ± 0.06 | 55.3 ± 0.01 | 7.1 ± 0.2 |
Insulin-loaded NPs after solvent evaporation | 520 ± 8 | 0.40 ± 0.02 | 0.25 ± 0.12 | 56.4 ± 0.03 | 8.7 ± 0.2 |
Sample | Concentration (µg/mL) | % of Viable HaCat MTT 12 h | % of Viable HaCat MTT 24 h |
---|---|---|---|
Commercial insulin solution in POLX gel | 2.5 | 100.0 ± 4.9 | 79.2 ± 7.2 |
20 | 90.1 ± 5.4 | 65.3 ± 2.9 | |
80 | 71.5 ± 5.6 | 52.7 ± 7.1 | |
Empty NPs in POLX gel | 2.5 | 96.8 ± 9.8 | 56.8 ± 8.7 |
20 80 | 87.2 ± 7.1 60.4 ± 3.6 | 55.2 ± 6.5 47.7 ± 4.8 | |
Insulin-loaded NPs in POLX gel | 2.5 | 93.3 ± 6.4 | 46.0 ± 6.1 |
20 80 | 69.7 ± 4.6 56.2 ± 9.1 | 39.4 ± 5.6 36.0 ± 1.7 |
Sample | Day 2 | Day 5 | Day 8 |
---|---|---|---|
Negative control (POLX gel) | | | |
Positive control (Commercial cream of silver sulfadiazine) | | | |
Commercial insulin solution in POLX gel | | | |
Insulin-loaded NPs in POLX gel | | | |
Samples | Epidermal Closure | Epidermal Differentiation | Amount of Granulation | Inflammatory Infiltration | Collagen Fiber Orientation | Pattern of Collagen | Total Score |
---|---|---|---|---|---|---|---|
Negative control (POLX gel) | 0.5 ± 0.4 | 1.0 ± 0.7 | 3.0 ± 0.7 | 2.5 ± 0.4 | 2.5 ± 0.4 | 2.5 ± 0.4 | 12.0 ± 2.8 |
Positive control (Commercial cream of silver sulfadiazine) | 0.7 ± 0.3 | 1.3 ± 0.5 | 3.3 ± 0.5 | 2.7 ± 0.3 | 2.3 ± 0.5 | 2.3 ± 0.5 | 12.7 ± 2.7 |
Commercial insulin solution in POLX gel | 0.8 ± 0.2 | 1.5 ± 0.4 | 3.5 ± 0.4 | 2.3 ± 0.2 | 2.8 ± 0.2 | 2.8 ± 0.2 | 13.5 ± 1.6 |
Insulin-loaded NPs in POLX gel | 0.8 ± 0.2 | 1.5 ± 0.4 | 3.5 ± 0.4 | 2.5 ± 0.3 | 2.8 ± 0.2 | 2.8 ± 0.2 | 13.8 ± 1.7 |
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Quitério, M.; Simões, S.; Ascenso, A.; Carvalheiro, M.; Leandro, A.P.; Correia, I.; Viana, A.S.; Faísca, P.; Ascensão, L.; Molpeceres, J.; et al. Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach. Int. J. Mol. Sci. 2021, 22, 4087. https://doi.org/10.3390/ijms22084087
Quitério M, Simões S, Ascenso A, Carvalheiro M, Leandro AP, Correia I, Viana AS, Faísca P, Ascensão L, Molpeceres J, et al. Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach. International Journal of Molecular Sciences. 2021; 22(8):4087. https://doi.org/10.3390/ijms22084087
Chicago/Turabian StyleQuitério, Maria, Sandra Simões, Andreia Ascenso, Manuela Carvalheiro, Ana Paula Leandro, Isabel Correia, Ana Silveira Viana, Pedro Faísca, Lia Ascensão, Jesús Molpeceres, and et al. 2021. "Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach" International Journal of Molecular Sciences 22, no. 8: 4087. https://doi.org/10.3390/ijms22084087
APA StyleQuitério, M., Simões, S., Ascenso, A., Carvalheiro, M., Leandro, A. P., Correia, I., Viana, A. S., Faísca, P., Ascensão, L., Molpeceres, J., Gaspar, M. M., & Reis, C. P. (2021). Development of a Topical Insulin Polymeric Nanoformulation for Skin Burn Regeneration: An Experimental Approach. International Journal of Molecular Sciences, 22(8), 4087. https://doi.org/10.3390/ijms22084087