Spatiotemporally Controlled Nuclear Translocation of Guests in Living Cells Using Caged Molecular Glues as Photoactivatable Tags

This method can help answer key question in the area of drug delivery, such as how to deliver a drug to a specific cellular-granular. The main advantage of this technique is that nuclear translocation of gas can be induced by light. Demonstrating the procedure will be Rina Mogaki and Akio Arisaka the grad students from my group.

To begin prepare the quantum dot linked cage molecular glue by synthesizing caged molecular glue linked dibenzocyclooctyne as described in more detail elsewhere. Prepare a 10 millimolar stock solution of the caged molecular glue linked dibenzocyclooctyne in dry DMSO. Then prepare the quantum dot linked caged molecular glue by first making aseye functionalized quantum dots.

To accomplish this add 100 microliters of 125 micromolar aseye PEG4 NHS ester in DMF to 400 microliters of a DMF solution containing 500 nanomolar of quantum dots that are coated with amine functionalized peg. Stir the mixture for one hour at room temperature. Place the resulting solution in a regenerated cellulose membrane and dialyze it for 24 hours against 800 ml of DMF.

Next dilute the stock solution of caged molecular glue linked dibenzocyclooctyne to 50 micromolar with DMF and add 200 microliters of it to the post dialysis solution. Stir the mixture for three hours at room temperature. Then dialyze the resulting mixture for 24 hours against 800 ml of fresh DMF using a regenerated cellulose membrane with the molecular weight cut off of 25000.

After 24 hours dilute the resulting solution to 200 nanomolar with DMF. Maintain human hepatocellular carcinoma HEP3 B-cells in Eagle's minimal essential medium containing 10%FBS under standard culture conditions. One day before the experiment seed 5000 HEP3 B-cells in 200 microliters of culture medium into each well of a 8 chambered glass slide.

Then cover the slide and place it into an incubator for 24 hours. The next day remove the culture medium and rinse the cells twice with 100 microliters of pre-warmed PBS. To each well of the 8 chambered slide add 200 microliters of FBS free medium containing 10 micromolar of a fluorescently dyed caged molecular glue.

After incubating for 3 hours remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. To visualize the endosomes add 200 microliters of culture media containing 100 nanomolar of an additional red fluorescent dye such as LysoTracker Red. Incubate the resulting cell sample for 20 minutes.

Then remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Afterwards return the cells to 200 microliters of the culture medium. To induce nuclear translocation of the fluorescently dyed caged molecular glue place the cells under a 100 watt xenon light source equipped with a 365 nanometer bandpass filter.

Expose the cells to UV light for 2 minutes. During this time keep the control cell sample without UV exposure in the dark. The lid of the glass substrates can be taken off for efficient UV exposure however take caution as prolonged exposure to UV light may cause cell death.

To visualize the nuclei add one microliter of Hoeschst stain to the culture medium. Incubate the resulting cell sample for 10 minutes. Then place the sample on the stage of a confocal laser scanning microscope.

Record the micrographs for the fluorescently dyed caged molecular glue, the LysoTracker Red, and the nuclear stain. As previously shown, seed 5000 human hepatocellular carcinoma HEP3 B-cells into each well of an 8 well chamber slide and culture under standard conditions for 24 hours. Supply the cell samples with 200 microliters of FBS free culture medium containing the fluorescently dyed caged molecular glue.

Incubate the resulting cell sample for 3 hours. Then remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Return the cells to 200 microliters of culture medium.

Then subject to sample confocal laser scanning microscopy and record the micrographs as described in the accompanying text protocol. As previously shown seed 5000 human hepatocellular carcinoma HEP3 B-cells into each well of an 8 well chamber slide and culture under standard conditions for 24 hours. Supply the cell samples with 200 microliters of FBS free culture medium containing 10 nanomolar of the quantum dot linked caged molecular glue.

Incubate the resulting cell sample for 3 hours. Then remove the medium and rinse the cell sample twice with 100 microliters of PBS. After rinsing the cells return them to 200 microliters of culture medium.

Then place the plate under a 100 watt xenon light source equipped with a 365 nanometer bandpass filter. Expose the cell sample to UV light for 2 minutes keeping the control samples in the dark. To visualize the nuclei add 1 microliter of Hoechst stain to the culture medium.

Incubate the resulting cell sample for 10 minutes. Then image the cells using a confocal laser scanning microscope. Seed the human hepatocellular carcinoma HEP3 B-cells the day before the experiment at 5000 cells per well into a 96 well culture plate.

Then cover the cells with 200 microliters of culture media and then place the plate in an incubator for 24 hours. The next day remove the culture medium and rinse the cell sample twice with 100 microliters of PBS. Then add 200 microliters of FBS free culture medium containing the fluorescently dyed caged molecular glue at concentrations ranging from 0.1 to 100 micromolar.

Incubate the plate for 3 hours and then place a plate under the UV light source. Expose the cell sample to UV light for 2 minutes keeping the control samples in the dark. Following UV exposure at 10 microliters of the Cell Counting Kit-8 reagent to the culture medium and incubate the resulting cell sample for 2 hours.

Then read the absorption of the samples at 450 nanometers using a microplate reader. Before photo radiation HEP3 B-cells incubated with fluorescently dyed caged molecular glue exhibits punctate fluorescence emission from the interior an analogous micrograph showing the LysoTracker Red indicates that fluorescently dyed caged molecular glue was localized in the endosomes. Accordingly the fluorescence emission assignable to the fluorescently dyed caged molecular glue was observed outside the cell nucleus prior to UV radiation.

After UV radiation the fluorescent suggests that fluorescently dyed caged molecular glue was uncaged and migrated into the cytoplasm and the cell nucleus. Such nuclear translation of the fluorescently dyed caged molecular glue can be induced site selectively by 2-photon near infrared light. The fluorescently dyed caged molecular glue in non irradiated areas did not escape from the endosomes and remained as punctate fluorescence.

The dendritic caged molecular glue tags can also deliver macromolecular guests such as quantum dots into the cell nucleus. These conjugates can be taken up into the cells and after UV exposure for 2 minutes the fluorescence emission of the quantum dots can be seen within the nucleus. After this development this technique paved the way for researchers in the field of gene therapy to explore low side effect treatment.