• Subscribe

Caught and caged: the future of drug delivery

Original truncated octahedron nanocage composed by twelve B-DNA helices forming the hexagonal faces and six square-shaped faces made up of four single-strand linkers. All images courtesy Mattia Falconi.

The DNANANO project is bringing the frontiers of nanotechnology to the heart of biomedicine. With innovative new designs for a more stable and effective breed of nanodevice, the emerging field of nano-biomedicine could soon welcome in a new era of targeted drug delivery.

In the current science and technology landscape, nanoscience promises huge advances for a variety of fields that are otherwise reaching their technological limitations. In the field of biomedicine, the potential of nanoscience to revolutionize targeted drug delivery is eagerly anticipated.

A major focus for nanoscience is in meeting the demand for new bottom-up methods of creating self-assembling nanodevices, which - thanks to strict properties of self-recognition - have their ideal ingredient in DNA. However, the routine use of synthetic oligonucleotides (short DNA or RNA molecules) and techniques such as cryogenic transmission electron microscopy (Cryo-TEM) and small-angle x-ray scattering (SAXS) make the construction and experimentation of 3D-DNA nanostructures a costly, low-yield procedure.

To reduce these expenses, the DNANANO project is running long-time atomistic simulations using the Curie supercomputer in France. By predicting the likelihood of successful self-assembly of DNA nanostructures and their structural properties, the costly trial-and-error element can be eliminated, ultimately allowing for more robust and accurate designs of DNA nanostructures.

Principally based at the University of Rome Tor Vergata in Italy (with the structural biology group), Federico Iacovelli has been using the Curie supercomputer to help create a potential drug delivery system by investigating a series of 3D-DNA 'nanocages'. Having ruled out any sequence combinations liable to give rise to unwanted structures, the nanocage simulations - obtained through classical molecular dynamics (MD) analysis - are screened before the DNA cages are experimentally assembled and subjected to spectroscopic analysis.

One square face of the polyhedron is modified by substituting four single-strand linkers with hairpins.

Seven million core hours on Curie - a PRACE (Partnership for Advanced Computing in Europe) tier-0 system - were awarded to the team under the PRACE 6th Call for Proposals. This was essential for the operations of the team, which is led by Mattia Falconi, also of the structural biology group at the University of Rome Tor Vergata. To run a MD simulation of a medium-sized nanocage for a single nanosecond on a normal workstation, one would need a spare 17 days. Performing a simulation in the region of 100 nanoseconds would take nearly a decade! The access to PRACE resources allowed the group to carry out multiple 100-nanoseconds simulations in less than thirty days.

Taking their cue from a nanocage published prior to DNANANO, Falconi and the team are looking to produce the first example of a preassembled DNA nanocage capable of a reversible, controlled encapsulation and release of cargo. The original nanocage is a truncated octahedron composed of twelve B-DNA helices forming the hexagonal faces and six square-shaped 'corners' made up of four single-strand linkers. "We are modifying one face of this polyhedron, one square face, by introducing hairpins," explains Falconi. In other words, the four single-strand linkers making up one of the corners are replaced with these hairpins, structures known to open when subjected to increasing temperatures.

At 37˚C (~99˚F), the original nanocage is unable to hold a cargo of the enzyme horseradish peroxidase (HRP), because its hexagonal faces are too small to afford the HRP entry. With its new hairpins, however, simulations of the modified nanocage at 37˚C show that it undergoes sufficient fluctuations and deformation to allow the HRP molecule access. Furthermore, at 4˚C (~39˚F) these effects are no longer observed, meaning that the introduction of hairpins effects a specific type of change whereby the protein can enter, reside, and leave the nanocage depending on the temperature. This is one of the first examples of a preassembled DNA nanocage capable of a reversible controlled encapsulation and release of its cargo. In a cellular context, this mechanism of encapsulation cannot feasibly be used in full for biomedical applications, but it does provide a remarkably strong proof-of-concept.

Temperature-controlled encapsulation and release of the enzyme horseradish peroxidase using a preassembled and covalently closed three-dimensional DNA nanocage structure as a controllable encapsulation device. As demonstrated, this design imposed a temperature-controlled conformational transition capability to the structure, which allowed entrance or release of an enzyme cargo at 37 °C while ensuring retainment of the cargo in the central cavity of the cage at 4 °C. Click above image for larger version.

Meanwhile, another series of similar octahedral nanocages has helped Falconi to gain important insights into the factors influencing their stability and dynamical properties. In three cages, the double helices are connected with different single-strand linkers - either thymidines, adenines, or a mixture of the two - to determine their impact. Surprisingly, classical MD techniques have shown that different linkers do little to alter the stability and dynamical properties of the nanocages, which can instead be attributed primarily to the geometry of the nanostructures themselves.

Though these nanocages are hardly affected by the differences between single-strand linkers, a change is altogether more apparent when they are replaced by double helices. Having experimentally assembled a nanocage entirely with double-strand linkers, MD analysis shows that the structure is much stronger and more stable than its single-strand predecessors.

Increasing stability brings with it the possibility of more robust designs. This wholly double-stranded DNA nanocage, for example, could potentially be employed as a building block for higher order structures.

This article is republished with permission from the PRACE 2014 Annual Report, which will be made available on the PRACE website next week. Be sure to read it in full to discover how PRACE is helping researchers to develop new particle accelerators, better understand combustion, and even explore the origins of our universe.

PRACE will also be holding its annual 'PRACEdays' event in Dublin, Ireland, from 26-28 May. Follow the hashtag #PRACEdays15 on social media to find out more about the event.

Although a solely temperature-stimulated nanocage carrier is not entirely practical, as a proof-of-concept it has provided the stimulus to further pursue what DNANANO started. "Now we are working on a new kind of mechanism for opening and closing our structure," explains Falconi. The idea is to introduce a programmable pH-triggered nano-switch into their nanocage by replacing two of the linkers in just one corner with two DNA triple helices, or with two mismatched hairpins. The pH-dependency of these replacement triple helices can be tweaked with precision to act as a gate, while the two mismatched hairpins take care of the temperature dependence.

With these controllable gates, it should be possible to produce a DNA nanocage that can transport and release its cargo under very specific pH and temperature conditions.

"We are collaborating with groups who are experts in DNA design, assembly, and spectroscopic characterization," states Falconi. "With colleagues from Tor Vergata's department of chemistry, we will investigate the pH we need to open the gates." He and his colleagues are now looking to use PRACE-assigned facilities once again, to provide them with a holistic understanding of the project and to gain insights that will allow a continual refinement of the proposed mechanism. If they can pull it off, this innovative DNA nanocage will mark a decisive step forward for nano-biomedicine.

Join the conversation

Do you have story ideas or something to contribute? Let us know!

Copyright © 2019 Science Node ™  |  Privacy Notice  |  Sitemap

Disclaimer: While Science Node ™ does its best to provide complete and up-to-date information, it does not warrant that the information is error-free and disclaims all liability with respect to results from the use of the information.


We encourage you to republish this article online and in print, it’s free under our creative commons attribution license, but please follow some simple guidelines:
  1. You have to credit our authors.
  2. You have to credit ScienceNode.org — where possible include our logo with a link back to the original article.
  3. You can simply run the first few lines of the article and then add: “Read the full article on ScienceNode.org” containing a link back to the original article.
  4. The easiest way to get the article on your site is to embed the code below.