Doxorubicin Loading and Release

The GI-Nc is capable of targeted chemotherapy by keeping doxorubicin (DOX) bound by the GI at physiological pH and releasing it in the acidic environment of endosomes. In these experiments, we verify the pH-dependent drug loading and release of our structure. 

DOX was loaded at pH 7.2 and pH 5.5 onto GI- and GcI-based duplexes, before using ethanol precipitation to isolate DNA-bound DOX from free DOX. These pH levels were used to simulate physiological pH and endosomal pH respectively. The amount of unbound DOX was determined via measurement of fluorescence.


Doxorubicin (DOX) is a common chemotherapy drug that works by intercalating into double-stranded DNA and inhibiting the progression of topoisomerase II [1]. As a result, it can be loaded onto our DNA duplexes with ease. In addition, DOX is auto-fluorescent (Ex/Em 488/570 nm), which allows for the determination of its concentration in solution through the use of fluorescent measurements [2].

The i-motif is a cytosine-rich DNA sequence that changes conformation based on the environmental pH. In acidic conditions, an anti-parallel tetramer structure forms through C-C+ Hoogsteen base pairing [3]. As a result, at a pH of 7.2, dox is securely bound to the GI-based duplex. However, when the pH decreases to 5.5 after endocytosis, the folding of the i-motif displaces the complementary strand and causes the release of the drug into the cell. Therefore, it is expected that less DOX would bind to the GI at pH 5.5 as compared to pH 7.2, but for the GcI-based duplex, the amount of bound DOX would not depend on the pH as there is no i-motif structure encoded.

Image 1: Mechanism of pH-dependent DOX loading and release through changes in i-motif conformation


To verify the pH-dependent loading and release of DOX by the duplex through the use of an i-motif sequence.

Techniques Used

Ethanol Precipitation of DNA

Ethanol precipitation is a method for concentrating and purifying DNA. It first involves the addition of cationic salts to neutralize the phosphate groups on the DNA backbone, which consequently decreases the polarity of the molecule. Afterwards, ethanol is added which causes the  DNA to precipitate out of the solution [4].


Image 2: Visual abstract of testing DOX loading and release

Sample Preparation

Doxorubicin (Sigma Aldrich) was diluted to a stock concentration of 10 mM. For this experiment, it was aliquoted and further diluted to a working concentration of 1000 μM. GI and GcI-based duplexes were diluted to two sets each of 0.25 µM, 0.5 µM, and 1 µM with pH 7.4 IDTE buffer (IDT) for a total of 16 samples. 

After adding DOX working stock to a final concentration of 20 µM in each sample, HCl was added to one set of samples to decrease the pH to 5.5. In summary, there were 8 samples (4 for GI and 4 for GcI) at pH 7.4 in IDTE and 8 samples (4 for GI and 4 for GcI) at pH 5.5. These samples were incubated for approximately 10 hours at 37°C.

Ethanol Precipitation

A salt solution with 2M NaCl and 0.1M MgCl2 was prepared in Milli-Q water, and 100% ethanol was chilled to -20°C. A volume of salt solution equivalent to 10% of the volume of the DOX-DNA solution was added to each sample and mixed thoroughly. Three times this volume of 100% ethanol was added, and the mixture was left to incubate at -20°C for one hour. 

After incubation, the samples were centrifuged at 12,000 rpm for 15 minutes at room temperature. The supernatant was extracted and loaded in triplicates into a 96-well plate to be read by plate reader.

Fluorescence Measurement

Fluorescence was measured at an excitation of 488 nm and an emission of 570 nm using a Varioskan plate reader.

Data Processing

After averaging the triplicate measurements and subtracting the absorbance of blank solutions (75% ethanol, 20% IDTE, 5% salt solution), the fluorescence measurements were expressed as a percentage of the fluorescence of a sample of 20 µM DOX with no DNA added. This amount was subtracted from 100 to yield the amount of DOX loaded.


Image 3: The percentage of DOX bound by GI- and GcI-based duplexes at pH 7.2 and 5.5 (A), with special emphasis on DOX binding by GI duplexes at different pHs (B) and the differential absorption of DOX by GI and GcI at pH 7.2 (C). Error bars represent standard deviation (n=3).

To showcase the behaviour of DOX at different pHs with different duplexes with clarity, the results from the samples containing 1 µM of DNA are presented in Image 3. There are two main features of graph A – the decrease in the amount of DOX loaded onto the GI-based duplexes and the increase in the amount of DOX loaded onto the GcI-based duplexes as the pH decreased (p<0.05, n=3). While the former demonstrates the i-motif’s role in inducing DOX release at pH 5.5, the latter is potentially a subject for further experimentation.

Graph B further emphasizes decreased DOX loading onto the GI with decreasing pH (p<0.05, n=3), and graph C showcases how the presence of the i-motif and its C-G base pair-rich regions enhance the ability of DOX binding for the GI as compared to the GcI at pH 7.2 (p<0.05, n=3). The graphs for all of the data points on the “Raw Data” spreadsheet linked above elucidates the same trend.


Based on the decrease in DOX loading onto the GI duplex at pH 5.5 compared to pH 7.2 and the inverse trend seen for GcI, it can be concluded that the presence of the i-motif interferes with DOX intercalation into DNA in acidic conditions. In order to determine the true amount of DOX that is able to be released by the GI under physiological conditions, further experimentation is required. DOX cannot diffuse out of the test tube as it could out of endosomes, so it may, to some extent, re-intercalate within the secondary structures of the GI strand.

  1. Cancer Research UK. (2017). Doxorubicin (Adriamycin). Retrieved 30 August 2019, from
  2. Motlagh, N. S., Parvin, P., Ghasemi, F., & Atyabi, F. (2016). Fluorescence properties of several chemotherapy drugs: doxorubicin, paclitaxel and bleomycin. Biomedical optics express, 7(6), 2400–2406. doi:10.1364/BOE.7.002400
  3. Abou Assi, H., Garavís, M., González, C., & Damha, M. J. (2018). i-Motif DNA: structural features and significance to cell biology. Nucleic acids research, 46(16), 8038–8056. doi:10.1093/nar/gky735
  4. Oswald, N. (2007). Ethanol Precipitation of DNA and RNA: How it works. Retrieved 30 July 2019, from,nucleic%20acids%20out%20of%20solution

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