Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction of the Expression Plasmid
2.2. Expression and Purification
2.3. HPLC Analysis
2.4. Stability of Fusion Proteins Test
2.5. Circular Dichroism Spectrometry
2.6. Cell Lines and Animals
2.7. Pharmacokinetics Analysis
2.8. MTT Assay
2.9. Colony Formation Assay
2.10. Cell Cycle Analysis
2.11. Migration Assay
2.12. Invasion Assay
2.13. Integrin Antibody-Blocking Assay
2.14. Western Blot Assay
2.15. Tumor Implantation and Treatment
2.16. Immunohistochemistry
2.17. Analytic Methods
3. Results
3.1. Expression and WB Identification of the Fusion Proteins
3.2. Purification and HPLC Analysis of the Fusion Proteins
3.3. Stability of Fusion Proteins
3.4. Circular Dichroism Spectral Analysis
3.5. Pharmacokinetic Analysis
3.6. HM-3/HSA Inhibited B16F10 Cells’ Viability
3.7. HM-3/HSA Inhibited B16F10 Cells’ Migration and Invasion
3.8. HM-3/HSA Bind to Integrin and Inhibited Integrin Signaling Pathways
3.9. HM-3/HSA Inhibited the Growth of the B16F10 Tumor
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
DMSO | dimethyl sulfoxide |
FBS | fetal bovine serum |
PBS | phosphate buffer saline |
HSA | human serum albumin |
MTT | 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide |
PCR | polymerase chain reaction |
CD | circular dichroism |
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Purification Step | HSA-HM-3 | HM-3-HSA | ||||
---|---|---|---|---|---|---|
Volume (mL) | Concentration (mg/L) | Recovery (%) | Volume (mL) | Concentration (mg/L) | Recovery (%) | |
Culture supernatant | 1000 | 205.5 | 100 | 1000 | 218.6 | 100 |
DEAE Sepharose 6 Fast Flow | 355 | 492.0 | 85.0 | 391 | 481.9 | 86.2 |
Phenyl Sepharose Fast Flow | 151 | 618.9 | 53.5 | 149 | 651.3 | 51.5 |
Parameter | HSA-HM-3 | HM-3-HSA |
---|---|---|
Alpha helix | 46.4 | 56.3 |
Parallel | 10.1 | 8.1 |
Beta-turn | 15.2 | 12.8 |
Random coil | 28.3 | 22.8 |
Parameter | HSA-HM-3 | HM-3-HSA |
---|---|---|
t1/2 (h) | 14.99 | 17.36 |
CLz (L/h/kg) | 140.23 | 156.62 |
AUC(0−t) (μg/L *h) | 127.99 | 110.43 |
AUC (0−∞) (μg/L *h) | 142.62 | 127.70 |
Vz (L/kg) | 3032.95 | 3923.60 |
Cmax (μg/L) | 5.99 | 7.09 |
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Li, T.; Zhang, H.-Z.; Ge, G.-F.; Yue, Z.-R.; Wang, R.-Y.; Zhang, Q.; Gu, Y.; Song, M.-J.; Li, W.-B.; Ma, M.-Z.; et al. Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities. Biomedicines 2021, 9, 1084. https://doi.org/10.3390/biomedicines9091084
Li T, Zhang H-Z, Ge G-F, Yue Z-R, Wang R-Y, Zhang Q, Gu Y, Song M-J, Li W-B, Ma M-Z, et al. Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities. Biomedicines. 2021; 9(9):1084. https://doi.org/10.3390/biomedicines9091084
Chicago/Turabian StyleLi, Ting, Han-Zi Zhang, Guang-Fei Ge, Zhao-Rong Yue, Ru-Yue Wang, Qian Zhang, Yan Gu, Mei-Juan Song, Wen-Bo Li, Min-Zhi Ma, and et al. 2021. "Albumin Fusion at the N-Terminus or C-Terminus of HM-3 Leads to Improved Pharmacokinetics and Bioactivities" Biomedicines 9, no. 9: 1084. https://doi.org/10.3390/biomedicines9091084