Generating a GI-Nc

(G-quadruplex, i-motif-Nanoclew)


Hodgkin’s Lymphoma

Hodgkin’s lymphoma (HL) is a type of cancer that arises from malignant mutations in B cells. B cells are lymphocytes, which are a type of immune cells that can produce antibodies, recognize antigens, and eliminate foreign particles [1]. HL is characterized by the presence of Reed-Sternberg cells, which are multinucleated and larger in size than healthy cells [2]. 

Since lymphocytes are found throughout the lymphatic system, HL can start anywhere in the body. Often, the disease starts within a group of lymph nodes, but rapidly metastasizes by migrating through the lymphatic system or bloodstream, rendering treatment particularly challenging [3].

Comparison of a normal lymphocyte to a Reed-Sternberg (Hodgkin’s Lymphoma) cell (National Cancer Institute)

Conventional Therapies

Cartoon of intravenous administration of chemotherapy


Chemotherapy is a systemic treatment in which drugs are introduced and carried throughout the body by the bloodstream [4]. Its mechanisms for eliminating rapidly dividing cancer cells include:

  • Damaging DNA to make the genome unreplicable
  • Mimicking nucleotides to disrupt replication and/or transcription
  • Binding to DNA to prevent the use of the genome by the cell

For HL, chemotherapy is used to treat all stages of the disease. It is given to destroy cancer cells, treat recurrences, and prepare the patient for stem cell transplants [5].

Limitations [6]
  • Nonspecifically targets all rapidly growing cells, including hematopoietic stem cells (progenitors of blood cells) and cells lining the digestive tract
  • Damages the cardiovascular, respiratory, and nervous systems
  • Induces fatigue, appetite loss, and susceptibility to infection

Photodynamic Therapy

Photodynamic therapy (PDT) for cancer treatment uses photosensitizers and light to induce cytotoxic effects [7]. After being injected into the bloodstream, photosensitizers are cleared out of healthy cells after 24 to 72 hours, but remain inside the malignant ones due to them chemically having a high affinity for tumor tissue [8]. An external light source is then used to incite reactive oxygen species generation, leading to cell death and/or blood vessel rupture, which depletes tumours of nutrients and oxygen [9].

While PDT is not yet a mainstream treatment for HL, it is effective in treating the endobronchial presentation of the disease [10] and has the potential to be effective for cutaneous (skin) manifestations [11].

Limitations [12]
  • Limited penetrance of tissue with some light sources
  • Causes scarring and inflammation of nearby healthy tissue
  • Exposure in the throat and lungs causes fluid build-up and hemoptysis
  • Cannot be used on cells around major blood vessels

Cartoon of patient undergoing photodynamic therapy


Scientific Interest

Targeted Drug Delivery

To combat the adverse side effects of conventional cancer therapies, targeted drug delivery devices such as antibody-drug conjugates, lipid-based nanoparticles, and gold nanoparticles, have been developed in addition to DNA-based structures. However, they face significant barriers to clinical translation, as described below.

Antibody-drug conjugates (ADC) are delivery systems composed of a monoclonal antibody, chemotherapy drugs, and a linker that holds the two together [13]. Its specificity comes from the ability of the antibody to target receptors found only on cancer cells.

Limitations [14]
  • The linker between the antibody and the drug degrades at a pH similar to that of the bloodstream, often causing premature release and off-target effects
  • Purification of ADCs from unconjugated antibodies is difficult, leading to competition for receptors between free antibodies and those loaded with drug
  • The number of common chemotherapy drugs suited to antibody conjugation, and the number of drug molecules able to be loaded per antibody are low

Cartoon of an ADC 

Cartoon of the cross-section of an LNP

Lipid-based nanoparticles (LNPs) include liposomes, bolaamphiphile aggregates, and solid lipid nanoparticles, which all rely on the formation of lipid membranes to enclose the drug [15]. Targeting to cancer cells can be passive (ie. not targeted to a specific receptor), or active via the use of aptamers or antibodies. For the former, the structures are localized to tumours due to the leaky vasculature-enhanced permeability and retention (EPR) effect [16].

Limitations [17]
  • Drug release hinges upon either external sources of heat and/or light, or requires the presence and activity of tumour-specific enzymes
  • The EPR effect is variable and depends on multiple factors, including the degree of tumour vascularization and the porosity of tumour vessels, which vary between cancers
  • Can induce system toxicity by triggering pro-inflammatory cytokine release, damaging liver tissues, and inducing hematologic toxicity

Gold nanoparticles (AuNPs) are gold cores coated with an organic monolayer. In addition to the conjugation of chemotherapeutics, AuNPs are also capable of photodynamic and photothermal therapies due to their chemical and optical properties [18]. They can target cancerous cells through the use of ligands or through passive accumulation in tumours via the EPR effect [19].

Limitations [20]
  • Although the AuNP core is non-toxic, the organic monolayer that is essential for stability, targeting, and drug delivery can be toxic depending on their chemical composition
  • Depending on the size of the particle, AuNPs may accumulate in healthy organs such as the liver, lungs, and brain, instead of in tumors 

Cartoon of the cross-section of an AuNP

Combinatorial Therapy

Cartoon symbolizing different pathways of therapeutic action in combinatorial therapy

Combinatorial therapy is the use of two or more therapies to target one disease. By targeting different pathways synergistically, combinatorial therapy reduces the risk of drug resistance while avoiding the time-consuming and costly process of novel drug development [21].

Limitations [22]
  • Unwanted side effects can be compounded if the therapies operate via similar mechanisms. Since identifying which agent is responsible for the increasing severity of symptoms is difficult, determining the appropriate dosage of each drug is challenging
  • Byproducts from the metabolism of one therapy may alter or inhibit the effects of the other

Technological Interest

We sought to make a drug delivery device that is biocompatible, actively targets cancer cells, and is triggered by environmental cues to execute multiple modalities of treatment. DNA has abundant therapeutic potential due to its programmability, which enables the creation of stable delivery vehicles and environmentally-responsive drug carriers. Two DNA structures are featured in our project: the i-motif and the G-quadruplex.

I-motifs are four-stranded DNA structures held together by hemi-protonated and intercalated cytosine base pairs. They typically undergo intra-strand folding under acidic conditions but can fold at other pHs with varying stability depending on the sequence and environmental factors [23].

Comparison between i-motif folding and Watson-Crick base pairing (Day et. al.)

Structure of G-quadruplex composed of stacked G-quartets (Rhodes et. al.)

G-quadruplexes are four-stranded DNA structures formed from runs of guanines separated by stretches of other base pairs. G-quartets, which are four guanines interacting with each other through cyclic Hoogsten hydrogen-bonding, are stacked via π-π interactions to make the quadruplex in the presence of cationic salts [24]. These non-covalent bonds allow for the binding of certain ligands, which include photosensitizers [25].

Our Aims

Taking into account all of the problems associated with existing targeted delivery and combinatorial systems, we sought to create, using only DNA, a drug delivery system that is:

  • Capable of active targeting
  • Responsive to the environment
  • Able to execute multiple modalities of treatment


Introducing the GI-Nc (G-quadruplex-i-motif-Nanoclew)

Our structure has three components – the nanoclew, the drug-loaded duplex, and the aptamer specific to HL cells. The nanoclew is a sphere composed of tightly coiled single-stranded DNA and made through a process called rolling circle amplification (RCA). This involves repetitively amplifying a circular template to make a long strand whose self-assembly into the nanoclew is directed by palindromic sequences [26]. 

The nanoclew is resistant to degradation by nucleases, in human serum, and at low concentrations [27]. Conjugated to it for stability are drug-loaded duplexes composed of the i-motif and G-quadruplex sequences, which deliver chemo- and photodynamic therapy respectively. Aptamers bound to the nanoclew allow for targeting to HL cells.

Cartoon of our complete structure

Cartoon of doxorubicin released from the DNA duplex

pH-dependent Release

Doxorubicin is an anthracycline that blocks topoisomerase II activity, leading to cell death. It intercalates into double-stranded DNA, enabling loading onto our entire duplex [28]. In the bloodstream at physiological pH, DOX is securely bound. However, after being endocytosed, the decrease in pH leads to a change in conformation of the i-motif and displacement of the complementary strand to release the drug intracellularly [29]. This ensures that DOX is only active within Hodgkin’s Lymphoma cells, which prevents off-target effects.

Photodynamic Therapy

In addition to chemotherapy, our structure is also capable of photodynamic therapy. Zinc phthalocyanine (ZnPC), a photosensitizer, has a large π-planar structure, which enables it to bind to the G-quadruplex via strong π-π stacking interactions [30]. Due to its poor solubility in aqueous environments, ZnPC requires the G-quadruplex as a carrier to generate significant amounts of reactive oxygen species [31]. As a result, our system enables ZnPC to induce cytotoxic effects after entering Hodgkin’s Lymphoma tumours in the lymph nodes and protects neighbouring cells from off-target, free photosynthesizer activity.

Cartoon of ZnPC loaded onto G-quadruplex and being irradiated

Cartoon of folded conformation of PS1NP aptamer 

Targeted Drug Delivery

Aptamers are DNA or RNA sequences that bind to specific targets. To target only Hodgkin’s Lymphoma cells with our therapeutic, we conjugated the PS1NP aptamer to our nanoclew base. The PS1NP aptamer was discovered through the Systematic Evolution of Ligands via Exponential Enrichment (SELEX) method, which is an in vitro procedure that iteratively sorts “binders” from “non-binders” from a large pool of unique sequences. It is reported to have a high affinity for HL cells and does not bind to any other type of blood cell [32].


Visual abstract showing the mechanism of the GI-Nc

To assemble the GI-Nc, DOX is loaded onto the duplex via intercalation into dsDNA, and ZnPC is loaded onto the G-quadruplex via π-π stacking interactions. The duplex is then annealed to the nanoclew for stabilization during transport. After entering the body, our structure binds to Hodgkin’s lymphoma cells using the PS1NP aptamer and consequently triggers endocytosis. As the endosome matures, the pH decreases, causing neighbouring i-motifs to interact with each other and fold. This change in conformation displaces the complementary strand, resulting in the release of doxorubicin. A 660 nm laser can simultaneously be used to activate photodynamic therapy by stimulating G-quadruplex-bound ZnPC localized in the lymph nodes.

Advancements Made

Here are the specific ways in which the GI-Nc improves upon existing devices, based on experiments in vitro:

Effective Active Targeting
Rapid Self-Assembly
High Stability
Combinatorial Therapy Potential
Low toxicity

Broader Implications

Beyond alleviating the problems associated with other drug delivery systems, the GI-Nc also has the following advantages for other avenues of research:

Versatility:  The Hodgkin’s-lymphoma-specific aptamer can be replaced with an aptamer for any other target, allowing the GI-Nc to theoretically treat any disease with a marker that can be targeted.

Size Tunability: The size of the nanoclew depends on the length of time for which rolling circle amplification proceeds, thus allowing the GI-Nc to vary in size depending on the user’s needs.

Novel Combinatorial Therapy Mechanism: As the GI-Nc is capable of executing both chemotherapy and photodynamic therapy, it provides the benefits of combinatorial therapy without an increase in the severity of side-effects associated with the use of multiple chemotherapy drugs. Side-effects are also minimized through drug encapsulation and targeted delivery.


Ideal Goal

The aim of our project is to create a therapeutic nanodevice capable of executing combinatorial therapy and verify its safety, specificity, and efficacy. This entails not only the creation of the structure and proof of its activity in vivo, but also, the use of other targeted drug delivery devices as controls. Due to time constraints, we set realistic goals to synthesize the nanoclew, anneal duplexes to it, evaluate drug loading and release in vitro, assess targeting and endocytosis of the structure, and confirm its cytotoxic effects on HL cells.

Project Timeline

  • Optimize circularization of the nanoclew template
  • Confirm nanoclew synthesis 
  • Determine the size and morphology of nanoclews
  • Determine the optimal conditions for duplex formation
  • Visualize the pH- and potassium ion-dependent activity of the i-motif and G-quadruplex
  • Assess annealing of duplexes to nanoclews
  • Examine the pH-dependent loading and release of doxorubicin
  • Verify the loading of ZnPC onto the G-quadruplex
  • Confirm generation of reactive oxygen species by irradiated ZnPC
  • Test binding affinity for and specificity to Hodgkin’s Lymphoma (HL) cells
  • Visualize endocytosis of our structure into HL cells
  • Assess the efficacy of the therapy on HL cells

Future Directions

  • Conjugation of aptamers to the structure for in vivo testing 
  • Optimization of the amount of DOX and ZnPC loaded for cell viability assays
  • Comparison of GI-Nc to conventional treatments (e.g. ZnPC by itself without a carrier, doxorubicin by itself without a carrier, other targeted drug delivery devices)

1. Canadian Cancer Society. (2019). The lymphatic system. Retrieved 23 July 2019, from

2, 3. Canadian Cancer Society. (2019). What is Hodgkin lymphoma?. Retrieved 23 July 2019, from

4. Chemotherapy – Canadian Cancer Society. (n.d.). Retrieved August 15, 2019, from

5. Chemotherapy for Hodgkin lymphoma – Canadian Cancer Society. (n.d.). Retrieved August 20, 2019, from

6. American Cancer Society. (2016). Chemotherapy Side Effects. Retrieved 23 July 2019, from

7, 9. Photodynamic Therapy for Cancer. (n.d.). Retrieved August 10, 2019, from

8. Nakajima, S., Hayashi, H., Omote, Y., Yamazaki, Y., Hirata, S., Maeda, T., … Sakata, I. (1990). The tumour-localizing properties of porphyrin derivatives. Journal of Photochemistry and Photobiology B: Biology, 7(2-4), 189-198.

10. Kiani, B., Magro, C. M., & Ross, P. (2003). Endobronchial presentation of Hodgkin lymphoma: a review of the literature. The Annals of Thoracic Surgery, 76(3), 967–972. doi: 10.1016/s0003-4975(03)00140-1

11. Photodynamic Therapy in Treating Patients With Lymphoma or Chronic Lymphocytic Leukemia – Full Text View. (n.d.). Retrieved August 11, 2019, from

12. Photodynamic Therapy. (n.d.). Retrieved August 11, 2019, from

13, 14. Nejadmoghaddam, M.R., Minai-Tehrani, A., Ghahremanzadeh, R., Mahmoudi, M.,Dinarvand, R., & Zarnani, A. (2019). Antibody-Drug Conjugates: Possibilities and Challenges. Avicenna J Med Biotechnol., 11(1), 3-23. 

15, 16. Puri, A., Loomis, K., Smith, B., Lee, J.-H., Yavlovich, A., Heldman, E., & Blumenthal, R. (2009). Lipid-Based Nanoparticles as Pharmaceutical Drug Carriers: From Concepts to Clinic. Critical Reviews in Therapeutic Drug Carrier Systems, 26(6), 523–580. doi: 10.1615/critrevtherdrugcarriersyst.v26.i6.10

17. Xue, H. Y., Liu, S., & Wong, H. L. (2014). Nanotoxicity: a key obstacle to clinical translation of siRNA-based nanomedicine. Nanomedicine, 9(2), 295–312. doi: 10.2217/nnm.13.204

18, 19. Arvizo, R., Bhattacharya, R., & Mukherjee, P. (2010). Gold nanoparticles: opportunities and challenges in nanomedicine. Expert Opinion on Drug Delivery, 7(6), 753–763. doi: 10.1517/17425241003777010

20. Sing, P., Pandit, S., Mokkapati, V.R.S.S., Garg, A., Ravikumar, V., & Mijakovic, I. Int J Mol Sci., 19(7), 1979. 

21. Mansoori, B., Mohammadi, A., Davudian, S., Shirjang, S., & Baradaran, B. (2017). The Different Mechanisms of Cancer Drug Resistance: A Brief Review. Advanced Pharmaceutical Bulletin, 7(3), 339–348. doi: 10.15171/apb.2017.041

22. Aumeeruddy, M. Z., & Mahomoodally, M. F. (2019). Combating breast cancer using combination therapy with 3 phytochemicals: Piperine, sulforaphane, and thymoquinone. Cancer, 125(10), 1600–1611. doi: 10.1002/cncr.32022

23. 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

24. Rhodes, D., & Lipps, H. J. (2015). G-quadruplexes and their regulatory roles in biology. Nucleic Acids Research, 43(18), 8627–8637. doi: 10.1093/nar/gkv862

25, 30. Yaku, H., Fujimoto, T., Murashima, T., Miyoshi, D., & Sugimoto, N. (2012). Phthalocyanines: a new class of G-quadruplex-ligands with many potential applications. Chem Commun, 48(50), 6203-6216. 

26. Ali, M.M., Li, F., Zhang, Z., Zhang, K., Kang, D., Ankrum, J.A., Le, C. & Zhao, W. (2014). Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine. Chem Soc Rev. 43(10), 3324-3341. DOI: 10.1039/C3CS60439J

27. Lv, Y., Hu, R., Zhu, G., Zhang, X., Mei, L., Liu, Q., Qiu, L., Wu, C., & Tan, W. (2015). Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers. Nature protocols. 10(10). 1508-1524. doi:10.1038/nprot.2015.078

28. Cancer Research UK. (2017). Doxorubicin (Adriamycin) | Cancer drugs | Cancer Research UK. Retrieved 23 July 2019, from

29. Park, H., Kim, J., Jung, S., Kim, W.J. (2017). DNA-Au Nanomachine Equipped with i-Motif and G-Quadruplex for Triple Combinatorial Anti-Tumor Therapy. Advanced Functional Materials, 28(5).

31. Matlou, G.G., Oluwole, D.O., Prinsloo, E., & Nyokong, T. (2018). Photodynamic therapy activity of zinc phthalocyanine linked to folic acid and magnetic nanoparticles. J Photochem Photobiol B., 186, 216-224. 10.1016/j.jphotobiol.2018.07.025.

32. Parekh, P., Kamble, S., Zhao, N., Zeng, Z., Wen, J., Yuan, B., & Zu, Y. (2013). Biostable ssDNA Aptamers Specific for Hodgkin Lymphoma. Sensors, 13(11), 14543–14557. doi: 10.3390/s131114543