We introduce the essential scientific background applied during our project.
DNA origami1 is a well-established technique to fold DNA into 2D or 3D shapes or patterns. It uses many short single strands of DNA, called “staple strands”, to fold a single long strand of DNA, the “scaffold strand”, into a preordained shape. The staple strands can be extended and conjugated with other materials, such as cholesterol, to enable the structure with additional capabilities. Furthermore, DNA origami structures naturally have a twisted conformation that can be mitigated using twist correction techniques in order to flatten the structure and avoid any adverse effects from an uneven surface.
In our project, we used DNA origami to create a base structure to attach the liposomes and gold nanoparticles. The DNA base ensures that the gold nanoparticles are in close proximity to the liposomes for the gold nanoparticle excitation to affect the liposome membrane.
Liposomes are artificial spherical structures commonly used as a drug delivery mechanism; the inside aqueous solution is able to retain both polar and charged molecules. The membrane itself is composed mainly of a lipid bilayer, a two-layered structure containing a hydrophilic phosphate head pointing outward towards an aqueous environment and a hydrophobic fatty acid tail oriented inwards. It was shown that by incorporating cholesterol in the composition of the bilayer, it would tighten the structure to reduce leakage; this discovery lead to the design and modification of liposomes.
For our project, we chose to use liposomes due to their capabilities to effectively carry drugs to the target area while have a triggered release mechanism. In addition, after releasing the drug, the liposome can reform allowing the body to absorb them for later use.
Gold nanoparticles (AuNPs)
Spherical gold nanoparticles measure anywhere from 2 to 400 nanometers in diameter and exist in various shapes such as solid or hollow spheres, rods, and urchin shapes. The charge of a gold nanoparticle, a useful property for electrostatic interactions, is determined by the synthesis process used to create them. AuNPs provided from Nanopartz use a PAH [Polyallylamine hydrochloride) surfactant and are made to a +25mV zeta potential by an industrial standard process. Gold’s excitable properties are explored as a triggering mechanism in our nanostructure.
Anchoring DNA origami structures to lipid nanoparticles
The creation of a compound liposome-DNA origami structure is based on a biologically inspired mechanism. By extending hydrophobic cholesterol-conjugated DNA staples from one side of our DNA origami structure, we create an amphipathic structure. The cholesterol then attracts and associates into the lipid bilayer membrane of an existing liposome, thereby anchoring the DNA origami structure to the liposome.
Cholesterol allows us to link the lipid bilayer to the DNA via a TEG linker. Binding the lipid bilayer to cholesterol is possible because of the hydrophilic and hydrophobic qualities of cholesterol.
Hydrophobicity and Hydrophilicity
Hydrophobicity (literally water-fearing) is the quality of a molecule that makes it repel water from itself. Hydrophilicity (literally water-loving) is the exact opposite where a molecule is attracted to water. These qualities are due to the polarity of a molecule. Some organic molecules such as cholesterol and phospholipids are composed of both hydrophilic regions and hydrophobic regions. Since hydrophobic and hydrophilic regions are attracted to regions of similar properties, cholesterol easily links with phospholipids.
Layering DNA origami with gold nanoparticles to join structures
We take advantage of DNA’s negatively charged phosphate backbone to electrostatically adhere positively charged gold nanoparticles onto the DNA origami surface. By controlling the stoichiometry of the reaction, we can use gold nanoparticles as a glue to join two LNP-DNA origami structures together.
Gold nanoparticle excitation and the triggered release of LNP
Once the structure is built, we can introduce near-infrared light to excite the gold nanoparticles to release heat. On the nanoscale, the gold nanoparticles will be in close enough proximity to adjacent liposomes such that the radius of generated heat will affect the stability of the liposomes. Once the liposome membrane is destabilized by the heat, its contents will be released to the external environment. The basis of our project relies on this final step to ensure accurate delivery of contents to the targeted location.
An absorbance spectrophotometer measures the absorbance of a sample over a range of wavelengths. Every compound has a unique absorption spectrum, and thus concentration can be measured relative to the absorbance of a particular wavelength. This relationship is described with Beer’s Law A=bcε. Where A is absorbance, b is pathlength, ε is molar absorptivity, and c is concentration2. Spectrophotometry can be used to detect the electrostatic interaction between AuNPs to the DNA origami structures. When binded together, we expect another peak to form on the graph, confirming the presence and yield of a gold-layered DNA origami structure3.
A fluorescence spectrophotometer measures the emission spectrum of a material in solution by exciting it at a set wavelength and measuring the light emitted from the relaxation of electrons to their ground state 22. This can be used to determine if certain compounds are present in the solution as each compound will have a different emission spectrum. We utilize the excitation wavelengths of the machine to test our triggering functions.
Gel Electrophoresis uses an electric field to separate a mixture of varying sizes of DNA fragments. The mixture of DNA fragments are placed in wells on one side of the setup and on the other side, a positive charge is applied which causes the negatively charged DNA to move towards the positive charge through the matrix/gel. This gel has a “sieving effect”; hence, different DNA fragments move different distances and allows us to separate our formed DNA structures from excess staples and scaffold.
Atomic Force Microscopy is utilized in non-contact mode for imaging in air for DNA origami. An atomically sharp tip, the “cantilever”, is placed very close to the sample surface and resonates. The device measures the deflection of a laser that shines onto the cantilever to render an image of nanoscale structures.
We use Cryo-electron Microscopy (Cryo-EM) to obtain a 2-Dimensional image of the final structure. The specimen is frozen into a thin sheet of ice and the scattering of an intersecting electron beam is measured. Based on the image we obtain, we can confirm whether the final structure has completely and correctly formed. In this case, a complete and correct structure would be a “sandwich” like structure with all the components (liposomes, DNA origami, and gold nanoparticles) present and in the right relative positions to each other4.
Ion exchange is the process of removing either cations or anions from a solution using electrostatic interaction. This is achieved by passing an ion-containing solution (mobile) through a solid phase. The solid phase is a material that contains ions which have a similar charge with the target ions in the aqueous phase which exchange through a reversible chemical reaction. The targeted ions are taken out of solution and stored in the solid phase. We use resin as our solid phase.
We use an anionic exchange column to facilitate the DNA and liposome anchoring process.
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Harris, D. (2010). Quantitative chemical analysis (8th ed.). New York: W.H. Freeman And Co. ↩
Sastry, M., Kumar, A., Datar, S., Dharmadhikari, C., & Ganesh, K. (2001). DNA-mediated electrostatic assembly of gold nanoparticles into linear arrays by a simple drop-coating procedure. Applied Physics Letters, 78(19), 2943-2945. doi:10.1063/1.1370993 ↩
Milne, J. L. S., Borgnia, M. J., Bartesaghi, A., Tran, E. E. H., Earl, L. A., Schauder, D. M.. . Subramaniam, S. (2013). Cryo‐electron microscopy – a primer for the non‐microscopist. FEBS Journal, 280(1), 28-45. doi:10.1111/febs.12078 ↩