Development and Characterization of Cationic Nanostructured Lipid Carriers as Drug Delivery Systems for miRNA-27a
Abstract
:1. Introduction
2. Results
2.1. Development of Nanostructured Lipid Carriers by High-Pressure Homogenization
2.2. Cytotoxicity of Nanostructured Lipid Carriers
2.3. Stability Studies of Nanostructured Lipid Carriers
2.4. Preparation of miRNA/cNLC Complexes
2.5. Complexation with Different Types of miRNAs
2.6. Morphology and Topographic Studies
2.7. Stability Studies of miRNA/cNLC Complex
2.8. Cytotoxicity Study
2.9. In Vitro Transfection and Differentiation of 3T3-L1 Cells
2.10. Cellular Uptake Studies
2.11. Confocal Laser Scanning Microscopy (CLSM)
3. Discussion
4. Materials and Methods
4.1. Materials
- double-stranded miRNA mimic mmu-miR-27a-3p (miRNA-27a) with the sequence UUC ACA GUG GCU AAG UUC CGC (MW 13,454 g/mol);
- miRNA mimic negative control (miRNA-NTC) with the sequence UCA CAA CCU CCU AGA AAG AGU AGA (MW 15,384.3 g/mol);
- miRNA mimic transfection control with fluorescence-labeled Cy3 group (FluoNTC) and the sequence UCA CAA CCU CCU AGA AAG AGU AGA (MW 15,891.5 g/mol).
4.2. Methods
4.2.1. High-Pressure Homogenization
4.2.2. Preparation of miRNA/cNLC Complexes
4.2.3. Particle Size and Zeta Potential Analysis
4.2.4. pH Measurements
4.2.5. Stability Studies
4.2.6. Entrapment Efficiency (EE%)
4.2.7. Agarose Gel Electrophoresis
4.2.8. Morphology and Topography Studies
4.2.9. Cell Viability and Cytotoxicity Studies (MTS and LDH Assay)
4.2.10. In Vitro Transfection and Differentiation Studies
Preparation of ORO Working Solution
4.2.11. Cellular Uptake Studies
4.2.12. Confocal Laser Scanning Microscopy (cLSM)
4.2.13. Statistical Analysis
5. 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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Formulation | PCS Data | LD Data (µm) | ELS Data | pH | |||
---|---|---|---|---|---|---|---|
z-ave (nm) | PdI | d(0.5) | d(0.9) | d(0.99) | ZP (mV) | ||
bNLC | 105.6 ± 1.8 | 0.180 ± 0.003 | 0.128 ± 0.001 | 0.189 ± 0.002 | 0.240 ± 0.001 | −18.2 ± 1.2 | 4.998 ± 0.039 |
cNLC–1 | 112.9 ± 1.7 | 0.185 ± 0.009 | 0.127 ± 0.001 | 0.188 ± 0.001 | 0.250 ± 0.002 | 14.6 ± 0.5 | 6.706 ± 0.047 |
cNLC–2 | 109.4 ± 1.4 | 0.213 ± 0.015 | 0.123 ± 0.001 | 0.187 ± 0.002 | 0.250 ± 0.001 | 33.9 ± 1.0 | 8.390 ± 0.081 |
cNLC–3 | 113.2 ± 0.8 | 0.213 ± 0.007 | 0.126 ± 0.004 | 0.184 ± 0.003 | 0.240 ± 0.013 | 39.6 ± 2.1 | 8.861 ± 0.053 |
cNLC–4 | 114.9 ± 1.7 | 0.207 ± 0.006 | 0.129 ± 0.001 | 0.188 ± 0.002 | 0.240 ± 0.003 | 40.6 ± 0.4 | 9.114 ± 0.021 |
cNLC–5 | 116.4 ± 2.8 | 0.217 ± 0.016 | 0.126 ± 0.002 | 0.186 ± 0.002 | 0.250 ± 0.004 | 43.0 ± 2.5 | 9.323 ± 0.001 |
cNLC–6 | 116.2 ± 3.7 | 0.218 ± 0.013 | 0.132 ± 0.002 | 0.183 ± 0.001 | 0.250 ± 0.009 | 46.8 ± 2.1 | 9.617 ± 0.014 |
Percentage of Reduction of Lipid Accumulation in Cells | miRNA/cNLC Complex | Control | |||||||
---|---|---|---|---|---|---|---|---|---|
1:2.5 | 1:5 | NTC | 27a | cNLC | Undif. Cells | ||||
NTC | 27a | NTC | 27a | ||||||
miRNA concentration | 25 nM | 10.77 ± 1.31% | 19.16 ± 1.56% | 3.11 ± 1.13% | 15.45 ± 1.36% | 0.77 ± 0.14% | 5.98 ± 0.42% | 12.87 ± 2.07% | 61.08 ± 3.75% |
50 nM | 1.01 ± 0.81% | 11.49 ± 1.52% | 1.53 ± 1.71% | 19.04 ± 0.86% | 1.17 ± 0.07% | 4.49 ± 0.07% | 12.39 ± 0.81% | ||
75 nM | 9.71 ± 0.58% | 20.20 ± 1.03% | 12.87 ± 1.21% | 24.37 ± 1.03% | 1.0 ± 0.35% | 6.58 ± 0.14% | 22.35± 0.51% | ||
100 nM | 11.08 ± 3.67% | 42.87 ± 2.11% | 34.71 ± 1.38% | 49.40 ± 0.97% | 7.48 ± 0.78% | 2.09 ± 0.21% | 37.9 ± 1.87% |
Formulation | Lipid Phase (%, w/w) | Water Phase (%, w/w) | ||||
---|---|---|---|---|---|---|
OA | Precirol® ATO 5 | Miglyol® 812 | Tween® 80 | Pluronic® F68 | MQ Water | |
bNLC | 0 | 4.50 | 0.50 | 1.33 | 0.67 | 93 |
cNLC–1 | 0.05 | 4.455 | 0.495 | 1.33 | 0.67 | 93 |
cNLC–2 | 0.10 | 4.41 | 0.490 | 1.33 | 0.67 | 93 |
cNLC–3 | 0.15 | 4.365 | 0.485 | 1.33 | 0.67 | 93 |
cNLC–4 | 0.20 | 4.32 | 0.480 | 1.33 | 0.67 | 93 |
cNLC–5 | 0.25 | 4.275 | 0.475 | 1.33 | 0.67 | 93 |
cNLC–6 | 0.50 | 4.05 | 0.450 | 1.33 | 0.67 | 93 |
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Tucak-Smajić, A.; Ruseska, I.; Letofsky-Papst, I.; Vranić, E.; Zimmer, A. Development and Characterization of Cationic Nanostructured Lipid Carriers as Drug Delivery Systems for miRNA-27a. Pharmaceuticals 2023, 16, 1007. https://doi.org/10.3390/ph16071007
Tucak-Smajić A, Ruseska I, Letofsky-Papst I, Vranić E, Zimmer A. Development and Characterization of Cationic Nanostructured Lipid Carriers as Drug Delivery Systems for miRNA-27a. Pharmaceuticals. 2023; 16(7):1007. https://doi.org/10.3390/ph16071007
Chicago/Turabian StyleTucak-Smajić, Amina, Ivana Ruseska, Ilse Letofsky-Papst, Edina Vranić, and Andreas Zimmer. 2023. "Development and Characterization of Cationic Nanostructured Lipid Carriers as Drug Delivery Systems for miRNA-27a" Pharmaceuticals 16, no. 7: 1007. https://doi.org/10.3390/ph16071007