PROJECT IDEAS

Generating a GI-Nc

Background

Hodgkin’s Lymphoma

Lymphocytes are immune cells that can produce antibodies, recognize antigens, and eliminate foreign particles. However, like any cell, abnormal growth can lead to the development of cancer. More specifically, B cells can mutate into Reed-Sternberg cells, which are larger than healthy lymphocytes and have more than one nuclei. This condition is a unique hallmark of Hodgkin’s Lymphoma.

Since lymphocytes are found throughout the lymphatic system, Hodgkin’s Lymphoma 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, causing serious challenges in its treatment.

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

Chemotherapy is a systemic therapy where drugs are introduced and carried throughout the body by the bloodstream. It eliminates rapidly dividing cancer cells by damaging DNA to make the genome unreplicable, mimicking nucleotides to disrupt replication and/or transcription, or binding to DNA to prevent its use by the cell.

For Hodgkin’s Lymphoma, chemotherapy is used to treat all stages of the disease. It is given to destroy cancer cells, treat recurrences, prepare the patient for a stem cell transplant, or control symptoms of advanced HL (called palliative chemotherapy).

Problems
  • Nonspecifically targets all rapidly growing cells, including hematopoietic stem 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) is a cancer therapy that uses photosensitizers and light to induce cytotoxic effects. After being injected into the bloodstream, photosensitizers are cleared out of healthy cells after 24 to 72 hours but remain inside cancerous ones. 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.

While PDT is not yet a mainstream treatment for Hodgkin’s Lymphoma, it is effective in treating the endobronchial presentation of the disease, and can potentially be effective for cutaneous manifestations.

Problems
  • Limited penetrance of 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

Relevance

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 all have significant problems.

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

Problems
  • 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 is low, and so is the amount of drug that can be loaded per antibody

Cartoon of an ADC [LABEL THE COMPONENTS?]

Cartoon of the EPR effect (or maybe we’ll find an image?)

Lipid-based nanoparticles include liposomes, bolaamphiphile aggregates, and solid lipid nanoparticles, which all rely on the formation of lipid membranes to enclose the drug. Currently, targeting to cancer cells is passive and the structures are localized to tumours due to the leaky vasculature-enhanced permeability and retention (EPR) effect.

Problems
  • Drug release hinges upon either external sources of heat and/or light, or requires the presence and activity of tumour-specific enzymes
  • Can only be used for cancers with solid tumours due to the necessity of the EPR effect for targeting – the use of antibody-coated liposomes has not yet achieved clinical success
  • 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 encapsulated within 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. They can target cancerous cells through the use of ligands, or through passive accumulation in tumours via the EPR effect.

Problems
  • Although AuNPs themselves are non-toxic, the functional ligands that enhance stability, carry drugs, and enable targeting can be harmful

Components of AuNPs (draw or find)

Combinatorial Therapy

An image related to 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.

Problems
  • Unwanted side effects can be compounded if the therapies operate via similar mechanisms, and identifying which agent is responsible for the increasing severity of symptoms is difficult
  • Byproducts from the metabolism of one therapy may alter or inhibit the effects of the other

Technological Interest

To make a superior targeted drug delivery device using nanotechnology, we sought to make all of our elements solely DNA-based, from creating a stable vehicle to a failsafe release mechanism that takes advantage of the macromolecule’s environmentally-dependent folding. DNA has abundant potential as a therapeutic due to its programmability and biocompatibility, and these two features are especially prominent in two secondary structures – the i-motif and the G-quadruplex.

I-motifs are four-stranded DNA structures held together by hemi-protonated and intercalated cytosine base pairs (C:C+). They typically fold under acidic conditions but can fold at other pHs with varying stability depending on the sequence and environmental factors.

Cartoon of folded i-motif

Cartoon of G-quadruplex 

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 pi-pi interactions to make the quadruplex in the presence of cationic salts.

Our Aim

Taking into account all of the problems associated with existing targeted delivery and combinatorial systems, we asked ourselves, “How do we create a drug delivery system that is capable of active targeting, responsive to the environment, and able to execute multiple modalities of treatment using only DNA?”

Merit

Introducing the GI-Nc

Our structure has three components – the nanoclew, the drug-loaded duplex, and the aptamer specific to Hodgkin’s Lymphoma 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. 

Conjugated to this stable base 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 Hodgkin’s Lymphoma cells.

Cartoon of our complete structure

Cartoon of doxorubicin bound to (top) and released from (bottom) the DNA duplex

pH-dependent Release

Doxorubicin is an anthracycline that blocks topoisomerase II activity, leading to cell death. Conveniently, it auto-intercalates into double-stranded DNA, enabling easy loading onto our duplex. 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. 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, a photosensitizer, is loaded onto the G-quadruplex portion of the duplex. Due to its poor solubility in aqueous environments, ZnPC requires the G-quadruplex as a carrier to generate significant amounts of reactive oxygen species. As a result, our system ensures that ZnPC induces 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

Folded conformation of PS1NP aptamer [CITATION]

Targeted Drug Delivery

Aptamers are “chemical antibodies” made of DNA or RNA 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 not bind to any other type of blood cell.

Mechanism

Visual abstract showing the mechanism of the GI-Nc

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 ZnPC in the lymph nodes.

Advancements Made

Here are the specific ways in which the GI-Nc improves upon existing devices:

GI-Nc Ab-Drug Conj Lipid NPs Gold NPs
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 be used to treat any disease with a unique marker.

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. 

Specification/Feasibility

Ideal Goal

The aim of our project is to create a therapeutic nanodevice capable of executing combinatorial therapy and prove 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 administrative and logistical issues, and time constraints, we instead set the following realistic goals for the competition.

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

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References

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

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

American Cancer Society. (2016). Chemotherapy Side Effects. Retrieved 23 July 2019, from https://www.cancer.org/treatment/treatments-and-side-effects/treatment-types/chemotherapy/chemotherapy-side-effects.html

American Cancer Society. (2018). Treating Classic Hodgkin Lymphoma, by Stage. Retrieved 23 July 2019, from https://www.cancer.org/cancer/hodgkin-lymphoma/treating/by-stage.html

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

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

Canadian Cancer Society. (2019). The lymphatic system. Retrieved 23 July 2019, from https://www.cancer.ca/en/cancer-information/cancer-101/what-is-cancer/the-lymphatic-system/?region=on

Canadian Cancer Society. (2019). What is Hodgkin lymphoma?. Retrieved 23 July 2019, from https://www.cancer.ca/en/cancer-information/cancer-type/hodgkin-lymphoma/hodgkin-lymphoma/?region=on

Canadian Cancer Society. (2019). The lymphatic system. Retrieved 23 July 2019, from https://www.cancer.ca/en/cancer-information/cancer-101/what-is-cancer/the-lymphatic-system/?region=on

Canadian Cancer Society. (2019). What is Hodgkin lymphoma?. Retrieved 23 July 2019, from https://www.cancer.ca/en/cancer-information/cancer-type/hodgkin-lymphoma/hodgkin-lymphoma/?region=on

Cancer Research UK. (2017). Doxorubicin (Adriamycin) | Cancer drugs | Cancer Research UK. Retrieved 23 July 2019, from https://www.cancerresearchuk.org/about-cancer/cancer-in-general/treatment/cancer-drugs/drugs/doxorubicin

Chemocare. (2019). Doxorubicin (Adriamycin, Rubex) Chemotherapy Drug Information. Retrieved 23 July 2019, from http://chemocare.com/chemotherapy/drug-info/doxorubicin.aspx

Chemotherapy – Canadian Cancer Society. (n.d.). Retrieved August 15, 2019, from https://www.cancer.ca/en/cancer-information/diagnosis-and-treatment/chemotherapy-and-other-drug-therapies/chemotherapy/?region=on.

Chemotherapy for Hodgkin lymphoma – Canadian Cancer Society. (n.d.). Retrieved August 20, 2019, from https://www.cancer.ca/en/cancer-information/cancer-type/hodgkin-lymphoma/treatment/chemotherapy/?region=on.

Dreaden, E. C., Austin, L. A., Mackey, M. A., & El-Sayed, M. A. (2012). Size matters: gold nanoparticles in targeted cancer drug delivery. Therapeutic Delivery, 3(4), 457–478. doi: 10.4155/tde.12.21

Gu, H., & Breaker, R.R. (2013). Production of single-stranded DNAs by self-cleavage of rolling circle amplification products. Biotechniques. 54(6). 337-343. DOI: 10.2144/000114009

Hodgkin Lymphoma – Cancer Stat Facts. (n.d.). Retrieved July 31, 2019, from https://seer.cancer.gov/statfacts/html/hodg.html.

Hodgkin lymphoma statistics – Canadian Cancer Society. (n.d.). Retrieved August 2, 2019, from https://www.cancer.ca/en/cancer-information/cancer-type/hodgkin-lymphoma/statistics/?region=on.

How We Started. (n.d.). Retrieved August 11, 2019, from https://www.ubcbiomod.com/2018/project-ideas/.

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

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

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

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

Photodynamic Therapy. (n.d.). Retrieved August 11, 2019, from https://www.cancer.org/treatment/treatments-and-side-effects/treatment-types/radiation/photodynamic-therapy.html.

Photodynamic Therapy for Cancer. (n.d.). Retrieved August 10, 2019, from https://www.cancer.gov/about-cancer/treatment/types/surgery/photodynamic-fact-sheet#r3.

Photodynamic Therapy in Treating Patients With Lymphoma or Chronic Lymphocytic Leukemia – Full Text View. (n.d.). Retrieved August 11, 2019, from https://clinicaltrials.gov/ct2/show/NCT00054171.

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

Quintanillad. (2015, November 16). Facts and Statistics. Retrieved August 2, 2019, from https://www.llscanada.org/disease-information/facts-and-statistics.

Reginato, E. (2014). Immune response after photodynamic therapy increases anti-cancer and anti-bacterial effects. World Journal of Immunology, 4(1), 1. doi: 10.5411/wji.v4.i1.1

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

Senapati, S., Mahanta, A., Kumar, S., & Maiti, P. (2018). Controlled drug delivery vehicles for cancer treatment and their performance. Signal Transduction And Targeted Therapy, 3(1). doi: 10.1038/s41392-017-0004-3

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