Zinc PC Loading + ROS Generation

To execute photodynamic therapy, ZnPC needs to be loaded onto the G-quadruplex region in order to be solubilized in vivo and transported into Hodgkin’s Lymphoma cells. After endocytosis, stimulation with a laser must effectively produce reactive oxygen species. 

In this experiment, we confirmed the loading of ZnPC onto the GI sequence through the use of scanning spectrophotometry. Following that, we performed a singlet oxygen sensor green assay to evaluate reactive oxygen production in response to laser irradiation.

Introduction

Zinc phthalocyanine (ZnPC), a photosensitizer, is commonly used in photodynamic therapy (PDT). This involves the use of light to induce cell death via the generation of reactive oxygen species. After ZnPC loads onto the G-quadruplex through 𝜋-stacking interactions, ZnPC-DNA complexes absorb light in the red spectrum due to a phenomenon called the bathochromic shift [1]. Therefore, if loading succeeded, we’d expect to see a peak in absorbance at 680 nm. 

Under physiological conditions, ZnPC is not soluble and instead forms aggregates that do not effectively generate reactive oxygen species (ROS) upon irradiation [2]. Therefore, a carrier is necessary for solubilization and induction of therapeutic effects. Based on this information, we’d expect greater ROS generation from bound ZnPC as compared to unbound.

Image 1: ZnPC loaded onto the G-quadruplex

Aims

To confirm the ability of ZnPC to be loaded onto the G-quadruplex

To evaluate reactive oxygen species generation by ZnPC after laser irradiation

Techniques Used

Scanning Spectrophotometry

Spectrophotometry is a tool that quantitatively analyses the property of materials depending on how much light is absorbed. Scanning spectrophotometry measures the absorbance of light at every wavelength within a range by comparing the difference in intensities between the beam emitted from the machine’s light source and the ray that hits the photodetector after passing through the sample [3].

Singlet Oxygen Sensor Green

Singlet Oxygen Sensor Green (SOSG) is a commercially available reagent composed of fluorescein and anthracene moieties. In its default state, the fluorescein is being constantly quenched by the anthracene. However, in the presence of reactive oxygen species, the anthracene forms an endoperoxide moiety and fluorescence occurs [4].

Methodology

Buffer Preparation:

Forty millimolar tris-acetate buffer was prepared by first dissolving Trizma acetate salt (Sigma Aldrich) in Milli-Q water. NaCl, KCl, and MgCl2 were added to bring the cation concentrations to 0.1M, 25 mM, and 10 mM respectively. DMSO was added to reach 10% v/v, and the pH was adjusted to 7.4 using acetic acid. 

Sample Preparation:

ZnPC (Sigma Aldrich) was dissolved in DMSO to a final concentration of 500 uM. GI sequences were solubilized in the buffer prepared above to a final concentration of 100 uM. Each sequence was then diluted to concentrations of 0 uM (only ZnPC), 2 uM, 10 uM, 20 uM, 50 uM, and 100 uM. The concentration of ZnPC in each sample was 5 uM. Fifty micromolar GI samples were also created without ZnPC to act as controls.

All samples were run on a UV-vis spectrophotometer (Cary 4000) from 250nm – 750nm using tris-acetate buffer as a blank.

Reactive Oxygen Species Generation:

Singlet Oxygen Sensor Green reagent (Thermo Fisher Scientific) was solubilized in methanol to make a 5 mM stock solution. This was then added to ZnPC or ZnPC and GI samples to a final concentration of 20 nM. A 650 nm laser was used to irradiate each sample for 2 minutes before measuring fluorescence at Ex/Em 504/525 nm using Varioskan.

Results

ZnPC Loading

Image 2: Graph of scanning spectrophotometry results for unbound ZnPC and ZnPC with different concentrations of GI sequence

As shown in Image 2, while ZnPC or GI sequence alone does not absorb at 680 nm, a clear peak forms when the two are combined. The height of this peak increases with increasing concentration of DNA, demonstrating that the more ZnPC binding sites (G-quadruplexes) there are and the more ZnPC that is bound, the greater the bathochromic shift.

Reactive Oxygen Species Release

Image 3: Graph of fluorescence from SOSG as a function of GI concentration

Image 3 demonstrates that the amount of ROS generated increases with the amount of G-quadruplex available to bind ZnPC, and that ZnPC alone barely yielded more fluorescence than the blank solution. While the amount of ROS generated increases sharply at lower concentrations, it does eventually plateau, signifying complete binding of all ZnPC. This is in accordance with our findings that more bound ZnPC results in greater absorbance at 680 nm, since absorbance of red laser radiation is necessary for reactive oxygen species generation.

Discussion

Overall, the data gathered supports the claim that ZnPC needs to be loaded onto the G-quadruplex to be effectively used for photodynamic therapy. This is demonstrated by how ZnPC alone does not absorb 680 nm light or efficiently generate ROS but readily does both when combined with the GI sequence. Furthermore, so far, increasing the amount of GI available for binding also increases the absorbance and improves ROS generation.

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