Tag Archives: Carl Walkey

Precision delivery of synthetic vaccines using DNA scaffolds

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When reading about nanomedicine, one is struck by the focus on precision especially with the regard to drug delivery and other therapeutics. There’s almost always a reference to repairing or destroying  malfunctioning/diseased tissue or cells to the exclusion of the  healthy tissues/cells.

The latest work from Arizona State University has raised a great deal of interest not just with this latest announcement but also some previous work. From the July 27, 2012 posting by Dexter Johnson on his Nanoclast blog on the IEEE [Institute for Electrical and Electronics Engineers],

About 18 months ago, the nanotech trade press was buzzing with the work of Hongbin Yu and Hao Yan, both from Arizona State University (ASU), when they developed a method that used DNA origami as a scaffold. When the DNA scaffolding was combined with “nano islands” made from gold, it enabled the manufacturing of smaller electronic memory devices.

Now [July 2012] Yan has joined with Yung Chang, a biodesign immunologist also from ASU, to use three-dimensional DNA structures as a scaffold on which they piggybacked synthetic vaccine complexes to make the delivery of the vaccines safer and more effective.

There are more details in the July 25, 2012 news item on ScienceDaily,

DNA nanotechnology, where the molecule of life can be assembled into 2-D and 3-D shapes, has an advantage of being a programmable system that can precisely organize molecules to mimic the actions of natural molecules in the body.

“We wanted to test several different sizes and shapes of DNA nanostructures and attach molecules to them to see if they could trigger an immune response,” said Yan, the Milton D. Glick Distinguished Chair in the Department of Chemistry and Biochemistry and researcher in Biodesign’s Center for Single Molecule Biophysics. With their biomimicry approach, the vaccine complexes they tested closely resembled natural viral particles in size and shape.

As proof of concept, they tethered onto separate pyramid-shaped and branched DNA structures a model immune stimulating protein called streptavidin (STV) and immune response boosting compound called an adjuvant (CpG oligo-deoxynucletides) to make their synthetic vaccine complexes.

First, the group had to prove that the target cells could gobble the nanostructures up. By attaching a light-emitting tracer molecule to the nanostructures, they found the nanostructures residing comfortably within the appropriate compartment of the cells and stable for several hours — -long enough to set in motion an immune cascade.

Next, in a mouse challenge, they targeted the delivery of their vaccine cargo to cells that are first responders in initiating an effective immune response, coordinating interaction of important components, such as: antigen presenting cells, including macrophages, dendritic cells and B cells. After the cargo is internalized in the cell, they are processed and “displayed” on the cell surface to T cells, white blood cells that play a central role in triggering a protective immune response. The T cells, in turn, assist B cells with producing antibodies against a target antigen.

To properly test all variables, they injected: 1) the full vaccine complex 2) STV (antigen) alone 3) the CpG (adjuvant) mixed with STV.

Over the course of 70 days, the group found that mice immunized with the full vaccine complex developed a more robust immune response up to 9-fold higher than the CpG mixed with STV. The pyramid (tetrahedral) shaped structure generated the greatest immune response. Not only was immune response to the vaccine complex specific and effective, but also safe, as the research team showed, using two independent methods, that no immune response triggered from introducing the DNA platform alone.

Here’s a little background information that may help to explain why researchers are looking for new ways to deliver vaccines, from the July 30, 2012 essay by Carl Walkey (University of Toronto) for the Nanowerk Spotlight series,

Traditionally, vaccines were formulated using attenuated or inactivated versions of the microbes they were intended to treat. However, inactivated microbes do not often elicit a strong enough immune response to induce antibody production. Attenuated viruses, on the other hand, may revert back to an active form within the body. There are also inherent difficulties in ensuring batch-to-batch consistency of the formulations. These shortcomings have led to a progressive shift towards the development of synthetic vaccines.

Synthetic vaccines can combine a portion of the target microbe, known as an ‘antigen’ together with an adjuvant that stimulates the immune system. They are more reproducible and have the potential to induce consistent and tailored immune responses. Yet, delivering both the adjuvant and antigen together to the appropriate immune cells is challenging.

While the developments at Arizona State University are exciting, it’s still a long way before there will be any treatments, from the Walkey essay,

Although the results from this study are encouraging, they represent only a step towards the ultimate goal of making DNA nanostructure-based vaccines a clinical reality. There are still many challenges.

“A big challenge from an immunological point of view is the stability of the particles” explains Chang. The body is equipped with an array of ‘nucleases’ – enzymes designed to degrade extracellular DNA. Nucleases may degrade the nanostructures before they reach their target.”

“I think safety will also be a major hurdle for the eventual clinical translation” he continues. “That will be the major concern people will have. It may cause an adverse effect or an auto-immune response. Those are the things we need to test thoroughly before moving into clinical trials.”

The researchers believe that the simplicity, robustness, and relative economy of the DNA nanostructures will be key advantages driving further development.

“DNA nanostructures have the advantage of self-assembling. You can produce them relatively simply with good reproducibility” says Yan. “With so many of the other nanoparticle systems, you have to synthesize different components chemically. This makes them difficult to scale-up.”

The July 24, 2012 news release from Arizona State University offers this comment on the potential,

Overall, though the field of DNA is still young, the research is advancing at a breakneck pace toward translational science that is making an impact on health care, electronics, and other applications.

While Chang and Yan agree that there is still much room to explore the manipulation and optimization of the nanotechnology, it also holds great promise.  “With this proof of concept, the range of antigens that we could use for synthetic vaccine develop is really unlimited,” said Chang.

I like the idea of more precise delivery of drugs and other therapies. Intuitively, it just makes sense that you want to focus on the diseased or destroyed tissues while preserving as much of the healthy ones as possible but I keep wondering if there might be a more subtle disease process at work. The problem may not lie in the diseased cells or tissues themselves but may originate in an entirely different part of the body. If you ever watch someone who’s walking awkwardly, you may notice the problem isn’t the foot placement; the real problem is in the hips. You are in fact examining the symptom rather than the problem. In which case, more precise application of various therapies will alleviate symptoms for a time while the disease process carries on.

Environment influences nanomaterial reactions to biological cells

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The discussion I’ve seen around nanomaterials and toxicological effects has largely centered on shapes, size, aggregate/agglomerate, etc. By contrast, Carl Walkey’s July 24, 2012 Nanowerk Spotlight essay focuses on nanomaterial surfaces, bare or coated with serum proteins (Note: I have removed links),

Biomolecule adsorption to nanomaterials is usually studied from physiological fluids with suspended biomolecules. Examples include blood serum/plasma, pulmonary surfactant, and synovial fluid. However, until now the amount of those molecules has not been considered relevant to the study. In a recent article appearing in ACS Nano (“Effects of the Presence or Absence of a Protein Corona on Silica Nanoparticle Uptake and Impact on Cells”), Drs. Anna Salvati, Kenneth Dawson, and their colleagues at the University College in Dublin, Ireland, show that if nanoparticles are exposed directly to cells in the absence of suspended biomolecules, the nanoparticles will extract biomolecules directly from cells themselves.

In their experiments, the team exposed silica nanoparticles to cells in two sets. One set was introduced into cell culture media that was supplemented with the usual concentration of fetal bovine serum, and the other into media that had no serum additives. They then incubated both sets of particles with a lung cancer cell line and measured particle uptake kinetics and cell adhesion. Nanoparticles treated under both conditions associated with cells. However, the particles that were incubated in media alone associated to a much greater extent than those that were first incubated in serum. This indicates that the affinity of the bare nanoparticle surface to the cell is much higher than the affinity of an equivalent surface that is coated with serum proteins. [emphasis mine] Similar observations are reported before for other systems, where it was also found that uptake under serum-free conditions is higher.

Moe specifically,

“When the nanomaterial is put in contact with a physiological environment, it is given a menu of possible biomolecules to adsorb” explains Dawson. “It will essentially go shopping for the biomolecules that it wants. Over time, it will exchange with the environment until it finds the things that it really likes most. If you don’t give it enough biomolecules in the form of serum, it will extract components from the cells themselves.”

The same silica nanoparticles exposed to cells in the two different conditions had different cellular responses as well. Most of the serum-coated particles were taken up within vesicles in the cell cytoplasm and produced no overt signs of toxicity. In contrast, the particles without a serum coating adhered to the cell surface to a greater extent, were present in vesicles, and some were also found free-floating in the cytoplasm. Exposure to particles in absence of serum significantly decreased cell viability and caused cells to take on a rounded morphology that is indicative of cell death. Dawson believes that cell death from uncoated particles is the result of strong interactions between the particle surface and the cell surface, which may damage the cell membrane, and/or initiate aberrant signaling cascades. When serum proteins are adsorbed to the nanoparticles, they ‘passivate’ the surface and limit direct nanomaterial-cell interactions.

When considering the early interactions of a nanomaterial with a cell, Dawson points out that one cannot think of the nanomaterial alone. Instead, one must think of the nanoparticle and its adsorbed biomolecules as a fundamental unit. [emphasis mine]

Most importantly,

Dawson believes that researchers must pay closer attention to the composition of the biomolecular environment surrounding the particles and cells when performing in vitro experiments. In other words, it is as important to consider the composition of the biomolecules in the media as it is to consider the chemical nature of the nanoparticle and the cell type. [emphasis mine]

“What’s absolutely clear is that depending on the type of dispersion that you make up, whether you add 10% serum or 20% serum, you get different levels of cell uptake” says Dawson. “Indeed, you get different levels of damage as well. It is therefore not meaningful to say that the nanoparticle is or is not toxic in that simplistic way. You can make a material toxic if you really want to make it toxic. You can make many materials damage cells simply because these have high surface energy. However, in a realistic physiological environment, part of the particle surface is covered and so that kind of damage would not be applicable.”

I encourage anyone who’s interested in nanotoxicology to read Walkey’s essay in full as I’ve excerpted only a portion.

BTW, Carl Walkey is a PhD graduate student at the University of Toronto and a member of the Integrated Nanotechnology & Biomedical Sciences Laboratory (INBS). I last mentioned Walkey in my July 12, 2012 posting about his Nanowerk Spotlight essay on nanotoxicology and animal studies.

Free the rats, mice, and zebrafish from the labs—replace them with in vitro assays to test nanomaterial toxcicity

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The July 9, 2012 Nanowerk Spotlight article by Carl Walkey (of the University of Toronto) focuses on research by Dr. André Nel and his coworkers at the California NanoSystems Institute (CNSI) and the University of California Los Angeles (UCLA) on replacing small animal model testing for nanomaterial toxicity with in vitro assays,

Currently, small animal models are the ‘gold standard’ for nanomaterial toxicity testing. In a typical assessment, researchers introduce a nanomaterial into a series of laboratory animals, generally rats or mice, or the ‘workhorse’ of toxicity testing – zebrafish (see: “High content screening of zebrafish greatly speeds up nanoparticle hazard assessment”). They then examine where the material accumulates, whether it is excreted or retained in the animal, and the effect it has on tissue and organ function. A detailed understanding often requires dozens of animals and can take many months to complete for a single formulation. The current infrastructure and funding for animal testing is insufficient to support the evaluation of all nanomaterials currently in existence, let alone those that will be developed in the near future. This is creating a growing deficit in our understanding of nanomaterial toxicity, which fuels public apprehension towards nanotechnology.

Dr. André Nel and his coworkers at the California NanoSystems Institute (CNSI) and the University of California Los Angeles (UCLA) are taking a fundamentally different approach to nanomaterial toxicity testing.

Nel believes that, under the right circumstances, resource-intensive animal experiments can be replaced with comparatively simple in vitro assays.  The in vitro assays are not only less costly, but they can also be performed using high throughput (HT) techniques. By using an in vitro HT screening approach, comprehensive toxicological testing of a nanomaterial can be performed in a matter of days. Rapid information gathering will allow stakeholders to make rational, informed decisions about nanomaterials during all phases of the development process, from design to deployment.

I’ve excerpted a brief description of Nel’s approach,

Rather than using in vitro systems as direct substitutes for the in vivo case, Nel is using a mechanistic approach to connect cellular responses to more complex biological responses, attempting to employ mechanisms that are engaged at both levels and reflective of specific nanomaterial properties.

“You need to align what you test at a cellular level with what you want to know at the in vivo” says Nel. “If oxidative stress at the cellular level is a key initiating element, then by screening for this outcome in cells you more are likely to yield something more predictive of the in vivo outcome. We can do a lot of our mechanistic work at an implementation level that allows development of predictive screening assays.”

By measuring many relevant mechanistic responses, and integrating the results, Nel believes that the in vivo behavior of a nanomaterial can be accurately predicted, provided that enough thinking goes into the devising the systems biology approach to safety assessment.

According to Walkey’s article, this approach could result in a ‘reverse’ nanomaterial development process,

Nel’s approach will influence not only the way in which nanomaterial toxicity is assessed, but also the way in which nanomaterials are developed. Currently, nanomaterials are designed to meet the need of a particular application. Toxicity is then evaluated retrospectively. Formulations that exhibit unacceptable toxicity at that point may be abandoned after a significant investment in development. Because Nel’s approach generates toxicity information much faster than traditional techniques, it will be possible to integrate toxicity during the design of a new nanomaterial. The proactive characterization of nanomaterial toxicity will provide feedback during the design process, producing formulations that maximize efficacy and minimize risk.

This is a very interesting article (illustrated with images and peppered with accessibly explanations of the issues) for anyone following the ‘nanomaterial toxicology’ story.