研究目的
To develop a photodynamic therapy nano agent using methylene blue-encapsulated liposomes for enhanced cellular uptake and ROS generation in breast cancer treatment.
研究成果
MB-liposomes were successfully developed with high stability, fast intracellular uptake, enhanced ROS generation, and no in vivo toxicity, making them a promising PDT nano agent for cancer therapy. The research confirms the potential of liposomes as effective drug carriers, but further optimizations are necessary.
研究不足
The dark cytotoxicity of MB-liposome solution should be improved. Further optimization is needed, such as lower toxicity drug choice or surface modification. The study is preliminary, and more in vivo experiments are required to confirm low toxicity and efficacy.
1：Experimental Design and Method Selection:
The study involved synthesizing MB-liposomes using a zwitterionic polymer-lipid for self-assembly, characterizing their properties, and evaluating their efficacy in photodynamic therapy through in vitro and in vivo tests. Methods included UV-Vis spectrophotometry, TEM, DLS, RNO test, DCFH-DA assay, MTT assay, live/dead staining, intracellular uptake assays, and in vivo toxicity assessments.
2：Sample Selection and Data Sources:
Breast cancer cells (4T1 cells) from ATCC were used for in vitro studies, and male ICR mice for in vivo toxicity tests. Materials were sourced from various suppliers as listed in the paper.
3：List of Experimental Equipment and Materials:
Equipment included rotary evaporation system (Rotavapor R30, Buchi), UV-Vis spectrophotometer, TEM, DLS instrument, fluorescence microscope, and dialysis membranes. Materials included DSPC, DPPE-PCB, MB, SDS, RNO, imidazole, DMEM-HG, FBS, PSA, trypsin-EDTA, DCFH-DA, Annexin V-FITC, PI, DiI, and others as specified.
4：Experimental Procedures and Operational Workflow:
Synthesis of MB-liposomes involved lipid film formation, hydration, extrusion, and dialysis. In vitro tests included ROS generation assays, cytotoxicity MTT assays, live/dead staining, intracellular uptake with DiI labeling, and cell death pathway identification. In vivo tests involved tail vein injection in mice and monitoring body weight over 14 days.
5：Data Analysis Methods:
Statistical analysis used ANOVA with p ≤ 0.05 for significance. Data are expressed as means ± standard deviation.
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DMEM-HG
SH30003.02
Thermo Hyclone
Cell culture medium for growing breast cancer cells (4T1) in vitro.
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DCFH-DA
D6883
Sigma-Aldrich
Fluorescence precursor used in DCFH-DA assay to detect intracellular reactive oxygen species.
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Rotary Evaporation System
Rotavapor R30
Buchi
Used for rotating the solution during liposome synthesis to dissolve lipids and form liposomes.
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DSPC
850365
Avanti
Lipid component used in the formation of liposomes for encapsulating methylene blue.
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Methylene Blue
A18174
Alfa-Aesar
Photosensitizer dye encapsulated in liposomes for photodynamic therapy to generate reactive oxygen species.
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SDS
230420250
Acros Organics
Used to break down liposomes and quantify methylene blue release in stability tests.
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RNO
D172405
Sigma
Chemical used in RNO test to measure reactive oxygen species generation by bleaching assay.
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Imidazole
I0250
Sigma
Used in RNO test to form imidazole endoperoxide for singlet oxygen detection.
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FBS
04-001-1A
Biological
Fetal bovine serum added to cell culture medium to support cell growth.
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PSA
03-033-1B
Biological
Antibiotic-antimycotic solution used in cell culture to prevent contamination.
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Trypsin-EDTA
03-051-5B
Biological
Used to detach cells from culture dishes for subculturing and experiments.
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Annexin V-FITC Fluorescence Microscopy Kit
550911
BD Pharmingen
Used to stain cells for identifying apoptosis by detecting phosphatidylserine inversion.
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Propidium Iodide Staining Solution
556463
BD Pharmingen
Used to stain dead cells or late apoptotic cells by binding to DNA.
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DiI
468495
Sigma
Fluorescent dye used to label liposomes for tracking intracellular uptake in cells.
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