Generating a GI-Nc

Background

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

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].

Cartoon of patient undergoing photodynamic therapy

Relevance

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.

Cartoon of an ADC 

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].

Cartoon of the cross-section of an AuNP

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].

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.

Comparison between i-motif folding and Watson-Crick base pairing (Day 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].

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

Merit

Cartoon of our complete structure

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.

Cartoon of ZnPC loaded onto G-quadruplex and being irradiated

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.

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

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.

Specification/Feasibility

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.

• 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)