Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1—7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.1.1. Solid Phase Peptide Synthesis (SPPS) Reagents
2.1.2. Advanced Spray Drying and Physicochemical Characterization Reagents
2.1.3. In Vitro Cell Culture Reagents
2.2. Methods
2.2.1. Solid Phase Peptide Synthesis (SPPS)
2.2.2. Computational Predictions of Physicochemical Parameters of Ang (1—7) Peptide and PNA5 Glycopeptide
2.2.3. Solubility Studies
2.2.4. Partitioning and LogP Experiments
2.2.5. HPLC for Solubility and LogP Experiments
2.2.6. Preparation of SD and co-SD Particles by Organic Solution Advanced Spray Drying in Closed-Mode
2.2.7. Scanning Electron Microscopy (SEM)
2.2.8. Particle Sizing and Size Distribution
2.2.9. X-ray Powder Diffraction (XRPD)
2.2.10. Differential Scanning Calorimetry (DSC)
2.2.11. Hot-Stage Microscopy (HSM) under Cross-Polarizers
2.2.12. Karl Fisher Titration (KFT)
2.2.13. Confocal Raman Spectroscopy (CRM)
2.2.14. Attenuated Total Reflectance (ATR)–Fourier-Transform Infrared (FTIR) Spectroscopy
2.2.15. In Vitro Aerosol Dispersion Performance
2.2.16. In Vitro 2D Cell Viability of Human Cells
2.2.17. In Vitro 2D Transepithelial Electrical Resistance (TEER) at the Air–Liquid Interface (ALI) of Human Lung Epithelial Cells
2.2.18. In Vitro Cell Viability of 3D Human Airway Epithelia at the Air–Liquid Interface (ALI)
2.2.19. In Vitro Transepithelial Electrical Resistance (TEER) of 3D Human Airway Epithelia at the ALI
3. Results
3.1. Solid Phase Peptide Synthesis (SPPS)
3.2. Computational Predictions of Physicochemical Parameters of Ang (1—7) Peptide and PNA5 Glycopeptide
3.3. Solubility
3.4. Partitioning Study (LogP)
3.5. Scanning Electron Microscopy (SEM)
3.6. X-ray Powder Diffraction (XRPD)
3.7. Differential Scanning Calorimetry (DSC)
3.8. HSM under Cross-Polarizer Lens
3.9. Karl Fisher Titration (KFT)
3.10. ATR-FTIR Spectroscopy
3.11. Confocal Raman Microspectroscopy (CRM)
3.12. In Vitro Aerosol Dispersion Performance
3.13. In Vitro 2D Cell Viability of Human Cells
3.14. In Vitro Cell Viability of 3D Human Airway Epithelia
3.15. In Vitro 2D TEER at the ALI
3.16. In Vitro TEER 3D Human Airway Epithelia at the Air–Liquid Interface (ALI)
4. Discussion
5. Conclusions
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Formulations (Powder Composition = Molar Ratio) | SD Ang (1—7) SD PNA5 co-SD Ang (1—7):Trehalose (25:75) co-SD PNA5:Trehalose (25:75) | co-SD PNA5:Trehalose (20:80) |
---|---|---|
Feed concentration in solvent (% w/v) | 0.125 | 2.5 |
Solvent | Methanol | Methanol |
Aspiration gas: ultra-high purity nitrogen gas (L/hour) | 670 (55 mm in height) | 670 (55 mm in height) |
Inlet temperature (°C) | 150 | 150 |
Outlet temperature (°C) | 47–53 | 60 |
Inert loop temperature (°C) | −15 | −15 |
Pump rate (%) | Low (25%) (7.5 mL/min) | Low (25%) (7.5 mL/min) and Medium (50%) (15 mL/min) |
Aspirator (%) | 100 (35 m3/h) | 100 (35 m3/h) |
Solvent | Solubility of Ang (1—7) mg/mL ± Std dev | Ang (1—7) LogS * | Ang (1—7) LogS Calculated by MOE | Ang (1—7) Solubility Category According to USP Definition | Solubility of PNA5 mg/mL ± Std dev | PNA5 LogS * | PNA5 LogS Calculated by MOE | PNA5 Solubility Category According to the USP Definition |
---|---|---|---|---|---|---|---|---|
H2O | 174.03 ± 0.19 | −0.71 | −0.82 | Freely soluble (FS) | 275.33 ± 2.31 | −0.58 | −0.53 | Freely soluble (FS) |
PBS | 165.38 ± 0.34 | −0.73 | - | Freely Soluble (FS) | 305.05 ± 0.22 | −0.54 | - | Freely soluble (FS) |
NS | 140.37 ± 0.32 | −0.81 | - | Freely soluble (FS) | 269.33 ± 1.53 | −0.59 | - | Freely soluble (FS) |
Methanol | 1.00 ± 0.005 | −2.95 | - | Slightly soluble (SS) | 0.34 ± 0.01 | −3.48 | - | Very slightly soluble (VSS) |
Ethanol | 0.160 ± 0.001 | −3.75 | - | Very slightly soluble (VSS) | 0.12 ± 0.003 | −3.94 | - | Very slightly soluble (VSS) |
Powder Composition (Molar Ratio) | Mean Size (μm) | Size Range (μm) |
---|---|---|
Raw Ang (1—7) | unmeasurable | - |
Raw PNA5 | unmeasurable | - |
SD-Ang (1—7) | 0.86 ± 0.42 | 0.31–2.62 |
SD-PNA5 | 0.78 ± 0.42 | 0.24–2.48 |
Co-SD Ang (1—7):Trehalose (25:75) | 0.56 ± 0.21 | 0.20–1.25 |
Co-SD PNA5:Trehalose (25:75) | 0.59 ± 0.35 | 0.10–1.72 |
Parameter | Raw Ang (1—7) | SD Ang (1—7) | Raw PNA5 | SD PNA5 | Raw Trehalose | SD Trehalose | co-SD Ang (1—7):Trehalose (25:75) | co-SD PNA5:Trehalose (25:75) |
---|---|---|---|---|---|---|---|---|
Tg onset (°C) | 165.67 ± 4.43 | 170.16 ± 2.54 | 162.3 ± 3.47 | 168.37 ± 1.96 | - | 37.53 ± 1.43 | 143.12 ± 2.21 | 155.19 ± 2.60 |
Tg mid (°C) | 169.13 ± 6.54 | 175.10 ± 2.90 | 167.45 ± 3.66 | 172.78 ± 1.33 | - | 41.45 ± 0.70 | 143.21 ± 2.13 | 155.26 ± 2.61 |
Tg End (°C) | 173.99 ± 0.09 | 180.1 ± 2.28 | 170.05 ± 3.85 | 174.45 ± 2.41 | - | 45.68 ± 1.63 | 143.28 ± 1.96 | 155.32 ± 2.54 |
∆Cp (J/g.) | 1.80 ± 0.40 | 1.57 ± 0.04 | 1.19 ± 0.10 | 1.47 ± 0.00 | - | 0.59 ± 0.05 | 0.70 ± 0.12 | 1.26 ± 0.10 |
Endotherm1 onset (°C) | 217.54 ± 11.06 | 219.81 ± 3.92 | 231.98 ± 7.43 | 220.52 ± 4.61 | 94.70 ± 0.08 | 145.81 ± 2.92 | 178.41 ± 2.35 | 169.42 ± 1.42 |
Endotherm1 peak (°C) | 218.45 ± 10.84 | 221.29 ± 3.93 | 232.45 ± 7.21 | 220.70 ± 4.47 | 95.85 ± 0.12 | 146.3 ± 2.57 | 185.82 ± 1.65 | 179.53 ± 0.86 |
∆H1(Enthalpy) J/g | 45.96 ± 5.34 | 41.05 ± 2.67 | 81.37 ± 20.28 | 52.54 ± 4.96 | 91.92 ± 7.79 | 3.39 ± 0.68 | 27.08 ± 1.96 | 8.88 ± 0.66 |
Endotherm2 onset (°C) | - | - | - | 141.32 ± 1.20 | 182.90 ± 4.60 | 209.65 ± 8.03 | 204.26 ± 7.15 | |
Endotherm2 peak (°C) | - | - | - | 141.01 ± 1.14 | 189.76 ± 3.88 | 213.28 ± 7.12 | 207.65 ± 8.25 | |
∆H2 (Enthalpy) J/g | - | - | - | 106.90 ± 6.15 | 20.66 ± 2.30 | 34.02 ± 9.16 | 58.76 ± 28.99 | |
Endotherm3 onset (°C) | - | - | - | 196.54 ± 1.10 | - | - | - | |
Endotherm3 peak (°C) | - | - | - | 204.24 ± 1.10 | - | - | - | |
∆H3 (Enthalpy) J/g | - | - | - | 118.33 ± 4.17 | - | - | - | |
Exotherm (TC) °C | - | - | - | - | 99.41 ± 0.82 | 114.85 ± 8.68 | 142.71 ± 1.04 |
System | Water % (w/w) |
---|---|
Raw Ang (1—7) | 4.03 ± 0.13 |
Raw PNA5 | 4.64 ± 0.18 |
Raw Trehalose | 8.55 ± 1.50 |
SD Trehalose | 4.18 ± 0.54 |
SD Ang (1—7) | 3.15 ± 0.13 |
SD PNA5 | 2.27 ± 0.13 |
Co-SD Ang (1-—7):Trehalose (25:75) | 2.13 ± 0.44 |
Co-SD PNA5:Trehalose (25:75) | 2.99 ± 0.92 |
Powder Formulation Composition (Molar Ratio) | Emitted Dose (ED) (%) | Fine Particle Fraction (FPF) (%) | Respirable Fraction (RF) (%) | MMAD (μm) | GSD |
---|---|---|---|---|---|
Raw Ang (1—7) | 100.74 ± 2.69 | 7.75 ± 0.44 | 60.75 ± 0.48 | 5.53 ± 0.91 | 7.06 ± 0.75 |
Raw PNA5 | 101.52 ± 5.91 | 3.33 ± 0.15 | 41.26 ± 0.76 | 9.01 ± 0.64 | 2.89 ± 0.21 |
SD Ang (1—7) | 79.80 ± 2.06 | 44.45 ± 3.85 | 97.35 ± 2.00 | 1.72 ± 0.03 | 1.76 ± 0.07 |
SD PNA5 | 64.45 ± 1.73 | 50.52 ± 8.18 | 96.93 ± 2.39 | 1.99 ± 0.06 | 1.97 ± 0.17 |
Co-SD Ang (1—7): Trehalose 25:75 | 83.70 ± 1.53 | 50.21 ± 1.58 | 95.71 ± 1.57 | 1.70 ± 0.08 | 1.81 ± 0.10 |
Co-SD PNA5: Trehalose 25:75 | 86.26 ± 0.22 | 32.49 ± 0.28 | 91.26 ± 0.31 | 2.09 ± 0.02 | 1.81 ± 0.03 |
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Alabsi, W.; Acosta, M.F.; Al-Obeidi, F.A.; Hay, M.; Polt, R.; Mansour, H.M. Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1—7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers. Pharmaceutics 2021, 13, 1278. https://doi.org/10.3390/pharmaceutics13081278
Alabsi W, Acosta MF, Al-Obeidi FA, Hay M, Polt R, Mansour HM. Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1—7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers. Pharmaceutics. 2021; 13(8):1278. https://doi.org/10.3390/pharmaceutics13081278
Chicago/Turabian StyleAlabsi, Wafaa, Maria F. Acosta, Fahad A. Al-Obeidi, Meredith Hay, Robin Polt, and Heidi M. Mansour. 2021. "Synthesis, Physicochemical Characterization, In Vitro 2D/3D Human Cell Culture, and In Vitro Aerosol Dispersion Performance of Advanced Spray Dried and Co-Spray Dried Angiotensin (1—7) Peptide and PNA5 with Trehalose as Microparticles/Nanoparticles for Targeted Respiratory Delivery as Dry Powder Inhalers" Pharmaceutics 13, no. 8: 1278. https://doi.org/10.3390/pharmaceutics13081278