Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel’s Physicochemical and Mechanical Properties
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials and Instrumentation
2.2. Screening of HA-PDPH Synthesis via EDC- and DMTMM-Activation
2.3. Synthesis and Characterization of HA-Derivates
- HASH: 1H-NMR (500 MHz, D2O): δ = 4.7–3.2 ppm (HA-backbone), 2.9 ppm (m, 2H), 2.8 ppm (m, 2H), 2.1 ppm (m, 3H). DS (SH) = 13–65%.
- HAmFU: 1H-NMR (500 MHz, D2O): δ = 6.3 ppm (m, 1H), 6.1 ppm (m, 1H), 4.7–3.2 ppm (HA-backbone), 2.3 ppm (m, 3 H), 2.1 ppm (m, 3 H). DS (mFU) = 13–61%.
- HADBCO via DBCO-amine: 1H-NMR (500 MHz, D2O): δ = 7.6 ppm (m, 8H), 5.2 ppm (m, 1H), 4.7–3.2 ppm (HA-backbone/DBCO-amine), 2.6 ppm (m, 1H), 2 ppm (m, 4H). DS (DBCO) = 13–20%.
- HADBCO via DBCO-sulfo-PEG4-amine: 1H-NMR (500 MHz, D2O): δ = 7.6 ppm (m, 8H), 5.2 ppm (m, 1 H), 4.7–3.2 ppm (HA-backbone/PEG-DBCO), 2.6 ppm (m, PEG), 2.0 ppm (m, 4H). DS (DBCO) = 4–35%.
2.4. Determination of the Gelation Time Based on Pipetting Studies
2.5. Microfluidic Device Fabrication
2.6. Microfluidic Preparation of HA-Microgels
2.7. Swelling Studies and Stability Tests of HA-Microgels
2.8. Thermal Stability Studies
2.9. FITC-Dextran Permeability Studies
2.10. Real-Time Deformability Cytometry
2.11. Non-Spherical HASH-Microgels
2.12. Synthesis of Trifunctional HA(mFU, biotin, DBCO)
- HA (mFU, biotin): 1H-NMR (500 MHz, D2O): δ = 6.3 ppm (m, 1H), 6.1 ppm (m, 1H), 4.7–2.5 ppm (HA-backbone/PEG-biotin), 2.3 ppm (m, 3H), 2.1 ppm (m, 5H), 1.4–1.9 ppm (m, 6H). DS (biotin) ≈ 9.5%.
- HA (mFU, biotin, DBCO): 1H-NMR (500 MHz, D2O): δ = 7.6 ppm (m, 8H), 6.3 ppm (m, 1H), 6.1 ppm (m, 1H), 5.2 ppm (m, 1H), 4.7–2.5 ppm (HA-backbone/PEG-biotin/PEG-DBCO), 2.3 ppm (m, 3H), 2.1 ppm (m, 5H), 1.4–1.9 ppm (m, 6H). DS (DBCO) ≈ 9%.
2.13. MF Preparation and Proof of Availability of Functional Moieties in Trifunctional HA-Microgels
3. Results
3.1. Synthesis and Characterization of HA-Derivates for Microgel Formation
3.2. Droplet Microfluidics-Assisted Fabrication of HA-Microgels
3.2.1. Gelation Properties of HA-Microgels Based on Bulk Pipetting Studies
3.2.2. Microfluidic Formation of Microgel Precursor Droplets
3.2.3. Preparation of HAmFU-Microgels via Diels–Alder [4 + 2] Cycloaddition
3.2.4. Preparation of HADBCO-Microgels via SPAAC
3.2.5. Preparation of HASH-Microgels via UV-Initiated Thiol–Ene Reaction
3.3. Characterization of HA-Microgel Porosities
3.3.1. HA-Microgel Swelling Properties and Stability in Aqueous Media
3.3.2. Thermal Stability Studies
3.3.3. FITC-Dextran Permeability Studies
3.3.4. Elasticity
3.4. Preparation of Non-Spherical HASH-Microgels
3.5. Trifunctional HA-Microgels
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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HA-Derivate | Coupling Reagent a | DS |
---|---|---|
HASH | PDPH (7.1 mg, 0.031 mmol); 0.125 Eq | 13% |
PDPH (14.2 mg, 0.062 mmol); 0.25 Eq | 24% | |
PDPH (28.4 mg, 0.124 mmol); 0.5 Eq | 42% | |
PDPH (56.9 mg, 0.248 mmol); 1 Eq | 65% | |
HAmFU | 5-methylfurfurylamine (3.4 mg, 0.031 mmol); 0.125 Eq | 13% |
5-methylfurfurylamine (6.9 mg, 0.062 mmol); 0.25 Eq | 27% | |
5-methylfurfurylamine (13.8 mg, 0.124 mmol); 0.5 Eq | 50% | |
5-methylfurfurylamine (27.6 mg, 0.248 mmol); 1 Eq | 61% | |
HADBCO | DBCO-amine (8.6 mg, 0.031 mmol); 0.125 Eq | 13% |
DBCO-amine (17.1 mg, 0.062 mmol); 0.25 Eq | 20% | |
DBCO-sulfo-PEG4-amine (16.2 mg, 0.031 mmol); 0.125 Eq | 4% | |
DBCO-sulfo-PEG4-amine (32.5 mg, 0.062 mmol); 0.25 Eq | 14% | |
DBCO-sulfo-PEG4-amine (64.9 mg, 0.124 mmol); 0.5 Eq | 23% | |
DBCO-sulfo-PEG4-amine (129.8 mg, 0.248 mmol); 1 Eq | 35% |
Sample Label | HA-Derivate b | PEG-Crosslinker b | Fluorescent Labeling |
---|---|---|---|
HAmFU−0.5 | 3.50% (w/v) HAmFU c | 2.09% (w/v) PEG-mal2 d | 0.4 µL Atto565-maleimide f |
HAmFU−0.75 | 3.50% (w/v) HAmFU c | 3.14% (w/v) PEG-mal2 e | 0.4 µL Atto565-maleimide f |
HASH−0.5 | 3.56% (w/v) HASH c | 2.51% (w/v) PEG-norb2 d | 0.4 µL Atto565-maleimide f |
HASH−0.75 | 3.56% (w/v) HASH c | 3.77% (w/v) PEG-norb2 e | 0.4 µL Atto565-maleimide f |
HADBCO−0.5 | 4.66% (w/v) HADBCO c | 2.09% (w/v) PEG-azide2 d | 0.4 µL Atto565-azide f |
HADBCO−0.75 | 4.66% (w/v) HADBCO c | 3.14% (w/v) PEG-azide2 e | 0.4 µL Atto565-azide f |
Sample Label | Gelation Time a |
---|---|
HAmFU−0.5 | 64 ± 1 min |
HAmFU−0.75 | 58 ± 2 min |
HASH−0.5 | ≤1 s |
HASH−0.75 | ≤1 s |
HADBCO−0.5 | 58 ± 3 s |
HADBCO−0.75 | 26 ± 2 s |
Sample Label | Eq of Crosslinker | Ddroplet a | Dmicrogel (H2O) a | Swelling (H2O) b | Dmicrogel (PBS) a | Swelling (PBS) b |
---|---|---|---|---|---|---|
HAmFU−0.5 | 0.5 Eq PEG-mal2 | 24.4 ± 0.3 µm | 45.0 ± 1.0 µm | 184.3 ± 4.1 | 29.2 ± 0.9 µm | 119.6 ± 3.7 |
HAmFU−0.75 | 0.75 Eq PEG-mal2 | 26.3 ± 0.3 µm | 39.7 ± 1.0 µm | 151.2 ± 3.8 | 28.8 ± 0.4 µm | 109.5 ± 1.6 |
HADBCO−0.5 | 0.5 Eq PEG-azide2 | 24.6 ± 0.4 µm | 45.1 ± 1.2 µm | 183.4 ± 4.9 | 29.8 ± 1.1 µm | 121.1 ± 4.6 |
HADBCO−0.75 | 0.75 Eq PEG-azide2 | 26.2 ± 0.7 µm | 39.7 ± 1.3 µm | 151.4 ± 4.9 | 28.0 ± 0.5 µm | 106.8 ± 1.9 |
HASH−0.5 | 0.5 Eq PEG-norb2 | 23.7 ± 0.5 µm | 28.9 ± 0.7 µm | 122.1 ± 3.1 | 24.2 ± 0.7 µm | 102.0 ± 3.2 |
HASH−0.75 | 0.75 Eq PEG-norb2 | 24.0 ± 0.7 µm | 28.5 ± 0.8 µm | 118.6 ± 3.4 | 26.1 ± 0.9 µm | 109.1 ± 3.1 |
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Heida, T.; Otto, O.; Biedenweg, D.; Hauck, N.; Thiele, J. Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel’s Physicochemical and Mechanical Properties. Polymers 2020, 12, 1760. https://doi.org/10.3390/polym12081760
Heida T, Otto O, Biedenweg D, Hauck N, Thiele J. Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel’s Physicochemical and Mechanical Properties. Polymers. 2020; 12(8):1760. https://doi.org/10.3390/polym12081760
Chicago/Turabian StyleHeida, Thomas, Oliver Otto, Doreen Biedenweg, Nicolas Hauck, and Julian Thiele. 2020. "Microfluidic Fabrication of Click Chemistry-Mediated Hyaluronic Acid Microgels: A Bottom-Up Material Guide to Tailor a Microgel’s Physicochemical and Mechanical Properties" Polymers 12, no. 8: 1760. https://doi.org/10.3390/polym12081760