研究目的
To develop a photoactivable prodrug conjugate using a hPSC-specific lectin for the selective elimination of tumorigenic human pluripotent stem cells to enhance the safety of regenerative medicine applications.
研究成果
The BC2Tama-DOXPCB conjugate effectively and selectively kills hPSCs upon UV activation without harming non-hPSCs, demonstrating a novel, controllable method for eliminating tumorigenic cells in regenerative medicine. This approach is the first of its kind using lectin-prodrug conjugates and holds promise for targeted delivery of other compounds.
研究不足
The study may have limitations in the efficiency of doxorubicin release upon UV exposure, potential incomplete internalization or binding in heterogeneous cell populations, and the need for optimization of UV exposure parameters to minimize effects on non-target cells. Application is currently in vitro and may require further validation in vivo.
1：Experimental Design and Method Selection:
Designed a fusion protein BC2Tama by fusing rBC2LCN lectin with Tamavidin for biotin binding, conjugated with doxorubicin-photocleavable biotin (DOXPCB) to create a photoactivable prodrug. Used UV light for activation to release doxorubicin intracellularly.
2：Sample Selection and Data Sources:
Used human induced pluripotent stem cells (201B7 hiPSCs), human fibroblasts, human mesenchymal stem cells, HepG2 liver cancer cells, and hiPSC-derived hepatocytes. Cells were cultured in specific media.
3：List of Experimental Equipment and Materials:
Included E. coli for protein expression, affinity chromatography columns, HPLC systems, cell culture plates, UV lamps, microscopes, flow cytometers, and various reagents like doxorubicin, biotin derivatives, and antibodies.
4：Experimental Procedures and Operational Workflow:
Expressed and purified BC2Tama in E. coli, synthesized and purified DOXPCB, conducted cell culture and viability assays, exposed cells to UV light, measured cytotoxicity using CCK-8 kit, and performed flow cytometry for mixed cell analysis.
5：Data Analysis Methods:
Analyzed data using SDSPAGE, HPLC, fluorescence detection, absorbance measurements at 450 nm for viability, and statistical analysis with mean ± SD from triplicates.
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Microscope
IX51
Olympus
Observing cell morphology
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UVP Blak-Ray XX-15L UV Bench Lamp
XX-15L
analytikjena
Providing 365 nm UV light for photoactivation
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L-fucose-Sepharose
Affinity chromatography for purifying BC2Tama protein
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BCA protein assay
Thermo Fisher Scientific
Measuring protein concentrations
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XV Pantera MP Gel
17%
DRC
Electrophoresis for analyzing protein purity
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Bio-Safe Coomassie G-250 Stain
Bio-Rad
Staining proteins in SDS-PAGE gels
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Doxorubicin hydrochloride
Sigma-Aldrich
Cytotoxic agent used in prodrug synthesis
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PC biotin-NHS ester
Click Chemistry Tools
Photocleavable biotin for conjugating with doxorubicin
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Luna C18(2) column
5 μm, 100 ?, 250 × 10 mm
Phenomenex
Reversed-phase HPLC for purifying DOXPCB
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mTeSR1
Veritas
Cell culture medium for hiPSCs
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Matrigel
Becton Dickinson
Coating for cell culture plates
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MesenPRO RS medium
Thermo Fisher Scientific
Cell culture medium for hFibs and hMSCs
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Vi-CELL Cell Viability Analyzer
Beckman Coulter
Counting cells and assessing viability
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TC20 Automated Cell Counter
Bio-Rad
Counting cells
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Cell Counting Kit-8
CCK-8
Dojindo Laboratories
Measuring cell viability based on dehydrogenase activity
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Anti-p53 antibody
AF1355
R&D systems
Blotting for p53 protein detection
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