Endocytosis Verification

The execution of combinatorial therapy by the GI-Nc requires it to be endocytosed into the cell. The release of doxorubicin depends on the decrease in pH in the endosome, and the generation of reactive oxygen species in a targeted manner relies on internalization into Hodgkin’s Lymphoma (HL) cells.

In this experiment, we demonstrate the ability of the aptamer-nanoclew complex to be endocytosed into KMH2 HL cells through the use of fluorescence microscopy.

Introduction

Endocytosis is the internalization of material by the cell’s plasma membrane. In the context of this experiment, receptor-mediated endocytosis is what is of interest to us. During receptor-mediated endocytosis, cell-surface receptors located in clathrin-coated pits are triggered by the binding of foreign material [1]. This consequently induces budding to form vesicles, which merge with early endosomes [1].

For the GI-Nc, it is expected that it will follow the same pathway, with the aptamer being the trigger for endocytosis.

Image 1: Diagram of receptor-mediated endocytosis [CITATION]

Aim

To demonstrate the ability of the GI-Nc to be endocytosed by HL cells

Techniques Used

Fluorescence Microscopy

Phenol-chloroform extraction is a liquid-liquid extraction technique for purifying DNA samples. While liquids partition into the lower organic phase and proteins are in the interphase, DNA can be isolated from the upper aqueous phase. As a result, we used this technique to purify our circularized template-primer complexes.

Methodology

Nanoclew Synthesis

Fluorescently tagged nanoclews were synthesized using the same rolling circle amplification reaction mix as described in “Sizing and Characterization”, but with the addition of Cy5-dUTP’s (Sigma Aldrich) to a final concentration of 0.048 mM. The reaction volume was 100 uL. Aptamers were conjugated to the nanoclews in accordance with the protocol for “Aptamer Binding Confirmation.”

Cell Sample Preparation

KMH2 cells were collected by centrifuging at 1500 rpm for 5 minutes and washing twice with PBS, and then resuspended to 5x10^5 cells per mL in complete media.  Cells were seeded in a 6-well plate (working volume is 2 mL per well) for a total of 1 million cells per well in triplicates. An entire reaction’s (100 uL) worth of aptamer-nanoclew complex was added to each well, and, after gentle rocking of the plate, the cells were incubated for three hours in the incubator.

After incubation, cells were pelleted and washed twice with PBS. Fixation was then done by resuspending the cells in 4% formaldehyde in PBS (Thermo Fisher) and incubating at room temperature for 15 minutes. After two washes with PBS, cells were mounted onto slides with Prolong Gold with DAPI (Thermo Fisher) and left to cure overnight on the bench. Coverslips were sealed the next morning with clear nail polish and imaged using the [MICROSCOPE]

Results

Image 3: Photo of an agarose gel showing the banding patterns made by template-primer complexes that have been subjected to different ligation conditions

As demonstrated by Image 3, while all conditions yielded successfully ligated templates, incubation for only 30 minutes at either temperature resulted in an excess of linear templates. Additionally, the band for one-hour incubation at room temperature is darker than the band for one-hour incubation at 37C. 

Discussion/Conclusion

To minimize the amount of linear template leftover and maximize the amount of circularized template, incubating samples at room temperature for one hour is the optimal protocol (marked by a star in Image 3). This is supported by the band denoting excess linear template being fainter compared to the 30-minute incubation samples, and the band for the ligated template being darker than the one present in the sample that had been incubated at 37C. 

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