研究目的
To perform a comparative analysis of Photosens, Holosens, and Phthalosens in terms of cellular uptake dynamics, intracellular distribution, and their dark and photoinduced toxicity on human tumor cell lines.
研究成果
Phthalosens and Holosens show superior photodynamic activity and cellular uptake compared to Photosens, attributed to their physicochemical properties such as amphiphilicity and charge. These findings suggest their potential for clinical applications in photodynamic therapy, with Phthalosens exhibiting the highest efficacy due to membrane localization and rapid uptake.
研究不足
The study is limited to in vitro conditions using specific cell lines, which may not fully represent in vivo or clinical scenarios. The photosensitizers' effects were tested at fixed concentrations and light doses, and potential variations in other cell types or conditions were not explored. The aggregation state or stability of photosensitizers in biological environments could affect results.
1：Experimental Design and Method Selection:
The study involved comparative in vitro analysis using cell culture models, flow cytometry for uptake dynamics, laser scanning microscopy for intracellular distribution, and MTT assay for toxicity assessment. Theoretical models include photodynamic therapy principles and lysosomotropic effects.
2：Sample Selection and Data Sources:
Human tumor cell lines T24 (transitional cell carcinoma of the urinary bladder) and SK-HEP-1 (human hepatic adenocarcinoma) were used, sourced from ATCC. Photosensitizers were synthesized by Federal State Unitary Enterprise 'State Scientific Centre NIOPIK', Russia.
3：List of Experimental Equipment and Materials:
Equipment includes Synergy MX Microplate Reader, FACSCalibur flow cytometer, LSM 510 Axiovert 200M META microscope, centrifuge 5810R, LED light source. Materials include photosensitizers (Photosens, Holosens, Phthalosens), cell culture media (MEM, fetal bovine serum), dyes (MitoTracker green, LysoTracker green, BODIPY FL C5-ceramide with BSA, CellMask), MTT reagent, DMSO, and buffers (PBS).
4：Experimental Procedures and Operational Workflow:
Cells were cultured, incubated with photosensitizers at specified concentrations and times, analyzed for uptake by flow cytometry, imaged for distribution by LSM, and assessed for viability using MTT assay after dark incubation or irradiation with LED light. Colocalization studies used organelle-specific dyes.
5：Data Analysis Methods:
Data were analyzed using GraphPad Prism 6 software for calculating LD50 and statistical comparisons. Flow cytometry data normalized by maximum signal; colocalization analyzed from fluorescence profiles.
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LSM 510 Axiovert 200M META
LSM 510 Axiovert 200M META
Carl Zeiss
Used for laser scanning microscopy to study intracellular distribution of photosensitizers.
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Synergy MX Microplate Reader
Synergy MX
BioTek
Used for obtaining absorption and fluorescence spectra of photosensitizers and measuring optical density in MTT assay.
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FACSCalibur
FACSCalibur
Becton Dickinson
Used for flow cytometry to analyze photosensitizer accumulation in cells.
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Centrifuge 5810R
5810R
Eppendorf
Used for centrifuging cells after washing.
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LED Light Source
Used for irradiation in photodynamic activity studies.
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MitoTracker Green
Thermo Fisher Scientific
Used for staining mitochondria in colocalization studies.
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LysoTracker Green
Thermo Fisher Scientific
Used for staining lysosomes in colocalization studies.
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BODIPY FL C5-ceramide with BSA
Thermo Fisher Scientific
Used for staining Golgi apparatus in colocalization studies.
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CellMask
Thermo Fisher Scientific
Used for staining cell membrane in colocalization studies.
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MTT
3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
Alfa Aesar
Used in MTT assay to assess cell viability.
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