This document summarizes a student project that aims to improve photodynamic therapy (PDT) for cancer treatment. The student hypothesizes that inducing hypoxic conditions in tumor cells will decrease the effectiveness of a photosensitizer and bioluminescent reagent in PDT. In experiments, the student cultures tumor cells under normal and hypoxic conditions with different PDT treatments and measures cell viability. The results show that bioluminescence was not an effective light source due to poor absorption overlap with the photosensitizer. Further work is needed to optimize photosensitizers and light sources to overcome limitations of conventional PDT.

1. A Novel Approach to
Improving Photodynamic Therapy
Through Analysis of the Effects of
Induced Hypoxia
and
Utilization of Bioluminescence
Shannen Prindle
Oakton High School

2. Background Information
 Cancer is the second leading cause of death in the
United States
 The most common forms of cancer treatment are
radiation and chemotherapy
 Both treatments have negative health side effects for
patients

3. Photodynamic Therapy (PDT)
 Relatively new form of cancer treatment
 Utilizes an external light source to activate the
photosensitizing agent
 Drawbacks to current technique
 External light source
• Cannot penetrate very deep into tumor mass
• Limited areas of application due to this
 Large tumors
• Increased presence of anoxic/hypoxic regions hinders the production
of reactive oxygen species
• Difficulty when it comes to the drug properly dispersing throughout
the whole tumor

4. PDT Techniques
 Researchers have been attempting to overcome these
individual drawbacks through various approaches
 External light source
 One novel technique involves the utilization of bioluminescent
reagents as a light source
 Eliminates issues with constrained penetration depth
 New problem arises due to low light intensity
 Large tumors
 Increased oxygen delivery to cells
 Provide patients with hyperbaric oxygen therapy before PDT
 Increases drug diffusion and reactive oxygen species production

5. Goal of Project
 Combine these two novel techniques together
 Oxygen is a key reactant in the bioluminescence reaction, therefore
conjoining the use of this approach with that of increasing oxygen
concentrations would then increase light production
 Allows for a cyclic process of development and improvement upon
PDT
 Developing a proof of concept for this technique would then allow for
the approach to be developed further in order to raise its
effectiveness to that of current PDT

6. Hypothesis
If the cells are cultured under hypoxic conditions, then the
overall effectiveness of the photosensitizer and
bioluminescent reagent will decrease in terms of increased
cell viability.

7. General Procedure
Initial
Experimentation
• Testing of the enzymatic hypoxia induction system (glucose oxidase
and catalase)
• Preparation of cell culture plates and stock solutions
• Ensure the main experimental groups are functioning properly
Final
Experimentation
• 231-Luc cells treated with all four experimental conditions
Regular PDT, photosensitizer control, PDT with bioluminescence
under hypoxia, PDT with bioluminescence under “normoxia”
• CCK assay performed to determine cell viability

8. Preparation
 MDA-MB-231-Luc cells plated in 6-well
plates
 Configuration for initial
experimentation shown in top right
picture, whereas final experimentation
is below that
 Stock solutions created and then wells
were treated accordingly
 1X concentration of glucose
oxidase/catalase system
 Methylene blue (photosensitizer)
concentrations at 0, 10, 25, 50, 100,
and 250µM
 Cell viability assay performed using
CCK assay kit

9. Main Experimental Conditions
 Regular PDT
 Methylene blue photosensitizer
 Irradiated under white light two times
 Photosensitizer control
 Methylene blue
 Left in total darkness
 PDT with bioluminescence under hypoxia
 Methylene blue, luciferin (bioluminescence), glucose oxidase and catalase (hypoxia
induction system)
 Covered with aluminum
 PDT with bioluminescence
 Methylene blue, luciferin
 Left in darkness and normal oxygen concentrations

10. Results: Initial Experimentation

11. Results: Regular PDT

12. Results: Photosensitizer Control

13. Results: PDT with Bioluminescence
under Hypoxia

14. Results: PDT with Bioluminescence

15. Conclusion
 Methylene blue (MB) was confirmed to act as an effective photosensitizer
when under normal oxygen concentrations and irradiated by an external
light source
 Recommended concentration of 25µM MB selected was too high for this
particular cell line, as cell viability decreased during MB control tests at
10µM and higher. Cells were also efficiently killed by 10µM MB during
normal PDT treatment.
 The trend in cell viability during the MB + GOX/CAT + Luciferin test was
nearly identical to that of the MB control.
 MB with luciferin yielded the same general data trend as the MB control
and MB with luciferin under hypoxic conditions
 Strongly indicates that the wavelength of light emitted by the
bioluminescent reaction and the absorption peak of the MB
photosensitizer were a suboptimal match

16. Absorption vs. Emission
 Rough sketch of absorption peak of MB (blue) compared to emission peak
of Luciferin (yellow)
 The overlap was most likely not sufficient due to the high concentrations
of MB and/or the low light production levels of bioluminescence

17. Further Research
 Improvements
 If using MB again, lower concentrations to below 10µM
 Choose/create new photosensitizer which has an absorption peak that more closely
correlates to the emission peak of luciferin
 Increased number of replicates performed for each experimental group and MB
concentration
 Perform additional control tests regarding the enzymatic hypoxia induction system
 Expansion
 Test this experiment in a wide variety of cell lines
 Progress to animal testing so as to incorporate variable of penetration depth
 Integrate photoimmunotherapy technique as well so as to overcome additional
drawbacks to PDT regarding healthy tissue damage

18. Acknowledgements
 Dr. Zaver Bhujwalla
 Dr. Paul Winnard
 Dr. Dmitri Artemov
 Dr. Grace Wang

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