- 作者: Yu-Wen Wu, Cheng-Chain Huang, Chun Austin Changou, Long-Sheng Lu, Hadi Goubran and Thierry Burnouf
- 作者服務機構: 1. Graduate Institute of Biomedical Materials and Tissue Engineering, College of Biomedical Engineering, Taipei Medical University, 250 Wu-Xing Street, Taipei, 11031, Taiwan 2. Graduate Institute of Translational Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3. The Ph.D. Program for Cancer Biology and Drug Discovery, Center for Translational Medicine, Taipei Medical University, Taipei, Taiwan 4. International PhD Program in Biomedical Engineering, College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan 5. Department of Radiation Oncology, Taipei Medical University Hospital, Taipei, Taiwan 6. Translational Laboratory, Department of Medical Research, Taipei Medical University Hospital, Taipei, Taiwan 7. International PhD Program in Cell Therapy and Regeneration Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 8. Saskatoon Cancer Centre and College of Medicine, University of Saskatchewan, Saskatchewan, Canada
- 中文摘要:
- 英文摘要:
Background
Human platelets (PLT) and PLT-extracellular vesicles (PEV) released upon thrombin activation express receptors that interact with tumour cells and, thus, can serve as a delivery platform of anti-cancer agents. Drug-loaded nanoparticles coated with PLT membranes were demonstrated to have improved targeting efficiency to tumours, but remain impractical for clinical translation. PLT and PEV targeted drug delivery vehicles should facilitate clinical developments if clinical-grade procedures can be developed.
Methods
PLT from therapeutic-grade PLT concentrate (PC; N > 50) were loaded with doxorubicin (DOX) and stored at − 80 °C (DOX-loaded PLT) with 6% dimethyl sulfoxide (cryopreserved DOX-loaded PLT). Surface markers and function of cryopreserved DOX-loaded PLT was confirmed by Western blot and thromboelastography, respectively. The morphology of fresh and cryopreserved naïve and DOX-loaded PLT was observed by scanning electron microscopy. The content of tissue factor-expressing cancer-derived extracellular vesicles (TF-EV) present in conditioned medium (CM) of breast cancer cells cultures was measured. The drug release by fresh and cryopreserved DOX-loaded PLT triggered by various pH and CM was determined by high performance liquid chromatography. The thrombin activated PEV was analyzed by nanoparticle tracking analysis. The cellular uptake of DOX from PLT was observed by deconvolution microscopy. The cytotoxicities of DOX-loaded PLT, cryopreserved DOX-loaded PLT, DOX and liposomal DOX on breast, lung and colon cancer cells were analyzed by CCK-8 assay.
Results
15~36 × 106 molecules of DOX could be loaded in each PLT within 3 to 9 days after collection. The characterization and bioreactivity of cryopreserved DOX-loaded PLT were preserved, as evidenced by (a) microscopic observations, (b) preservation of important PLT membrane markers CD41, CD61, protease activated receptor-1, (c) functional activity, (d) reactivity to TF-EV, and (e) efficient generation of PEV upon thrombin activation. The transfer of DOX from cryopreserved PLT to cancer cells was achieved within 90 min, and stimulated by TF-EV and low pH. The cryopreserved DOX-loaded PLT formulation was 7~23-times more toxic to three cancer cells than liposomal DOX.
Conclusions
Cryopreserved DOX-loaded PLT can be prepared under clinically compliant conditions preserving the membrane functionality for anti-cancer therapy. These findings open perspectives for translational applications of PLT-based drug delivery systems. - 中文關鍵字:
- 英文關鍵字: Cryopreserved platelet, Tissue factor, Cancer, Doxorubicin, Platelet extracellular vesicles, Drug delivery