Introduction

In our project, we have used synthetic biology and engineering cycles to optimize nanoparticle production for photothermal therapy (PTT). In order to ensure that our project was in line with safety regulations, we consulted safety officers and managers from the Leiden Institute of Biology. They provided us with a risk assessment, which highlighted the 4S rule often implemented when determining the hazards associated while working with nanoparticles. The 4S rule focuses on the following factors: Structure (metal composition), Size (1-100 nm), Shape (e.g, fibers or spheres), and Surface (coating or surface area), which combined determine the toxic effect of the nanoparticle. In addition, we focused on safety while working with GMOs, and establishing a cell free system for biosafety purposes.

Below we provided the safety assessment that played a crucial role throughout our project. This document has been provided by the safety officers from Leiden University.

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All in all, we learned about the different processes and the safety aspects that come with producing and utilizing nanoparticles. On the page below you can read how we implemented various safety aspects during biological production of nanoparticles. Encounter some experiences we have had, and use this as a guide to make your future nanoparticle project safer!

Which material is safest to use?

Nanoparticles can be made of a variety of materials. The material choice should be in line with the favored application. We have explored the aspects of silver and gold nanoparticles in our literature review and we have chosen a combination of a silver core with golden spikes.

Definition of H-sentences:

H-sentences also referred to as Hazard Statements (H-codes), provide information about the hazard associated with a certain chemical. For example: Physical hazards. H200: Unstable explosive.

Gold nanoparticles and gold salts

Gold nanoparticles have received much interest in the field of biomedicine due to their electrical, mechanical, thermal, chemical, and optical properties. Despite several advantages, there are potential consequences when working with gold nanoparticles. These particles can interact with biological tissue and molecules, which risks severe toxicity1. Nanoparticles can have different cellular interactions depending on their size: if the nanoparticles are around 10 nm they can enter the nucleus and disrupt DNA-related processes2. Larger nanoparticles can also enter cells and disrupt cellular processes, such as actin organization2.

In our project, we used the salt gold chloride (HAuCl4) for the formation of golden spikes on the surface of bimetallic nanoparticles. Gold chloride is a reactive chemical with the following H-sentences: H290 (May be corrosive to metals), H302 (Harmful if swallowed), H314 (Causes severe skin burns and eye damage), H373 (May cause damage to organs through prolonged or repeated exposure), and H411 (Toxic to aquatic life with long-lasting effects)

Read under the what we did section to see the safety measures we took whilst working with these chemicals.

Silver nanoparticles and silver salts

Silver is widely used for medicinal purposes and in packaging due to its antibacterial activity and lower toxicity compared to other bactericides3. The toxicity of silver, including nanoparticles of silver, to humans is generally low3. Due to their lower reactivity silver nanoparticles are less dangerous to work with than their golden counterparts, for example. However, the toxicity is dependent on morphology and size4.

We used silver nitrate (AgNO3) for our nanoparticle synthesis. This salt has the following H-sentences: H272 (May intensify fire; oxidizer), H290 (May be corrosive to metals), H314 (Causes severe skin burns and eye damage), and H410 (Very toxic to aquatic life with long-lasting effects).

Read under the what we did section to see the safety measures we took whilst working with these chemicals.

Platinum nanoparticles

Although research suggests that platinum nanoparticles may also be ideal for PTT, we refrain from using it during our project due to some risks associated with it. Read more on the safety aspects we considered in the dropdown below.

Platinum nanoparticles (nano platinum) have been widely used in industry, medicine, and diagnostics. However, there are several reports related to toxic effects of nano platinum.

Animals intratracheally exposed to platinum nanoparticles have shown increased levels of proinflammatory cytokines. This was further checked with a bronchoscope which confirmed an inflammatory response in the lungs5. Oral administration of platinum nanoparticles can also cause an inflammatory response and induce oxidative stress5. Furthermore, nano platinum has also been found to induce hepatotoxicity and nephrotoxicity when intravenously introduced5.

Platinum nanoparticles can have promising applications, but due to toxicity and possible dangerous health effects, we would highly discourage an iGEM team to use platinum for nanoparticle synthesis.

What we did

Whilst working with the heavy metals we took several safety measures. We made sure to wear lab coats, nitrile gloves, and safety glasses. Gold and silver stock solutions (usually 100 mM) were made in a fume hood. When not in use, the gold and silver stock solutions were stored in a chemical cabinet.

Deciding on a safe size range and morphology.

Besides the material, the morphology plays a huge role in the intrinsic properties of nanoparticles. For example, a nanorod will react very differently to a laser compared to an urchin-like nanoparticle (see our MSP literature review on the Collaborations page).

Safety and size

The nanoparticle size and morphology play an important role as these largely determine the unique mechanism of nanoparticle interaction with living systems6. Accumulation of nanoparticles at sites outside of tumor tissue is undesirable since this can have cytotoxic effects. Investigations show that nanoparticles of 50 nm in size have a low organ accumulation in the spleen and liver8. Also, large nanoparticles greater than 250 nm are recognized by specific defense systems of the body and phagocytosed by the mononuclear phagocytes, which prevents them from entering other tissues6. Read more on our Project Description page about the size of nanoparticles.

What we did

We decided to generate a product that is efficient and safe for PTT, thus we aimed to produce nanoparticles at a size of 20-150 nm.

Working with GMOs

During our project, we overexpressed several genes from metal reduction pathways in E. coli to enhance and optimize nanoparticle synthesis. We have also used pre-existing E. coli overexpression libraries (ASKA) to see the effect of these genes.

ASKA collection:

The ASKA collection (A Complete Set of Escherichia coli K-12 ORF Archive) library, which comprises every E. coli ORF cloned into the expression vector pCA24N

Bacteria

We provide a short description on how the GMO would affect nanoparticle synthesis and the safety factors to consider when making GMOs.

The type of bacterial species you would use for nanoparticle synthesis will highly influence your final product. Several microorganisms are known to trap metals in situ and convert them into elemental nanoparticle forms. Other reports suggest that nanotization is a way of stress response and biodefense mechanism for the microbe, which involves metal excretion/accumulation across membranes, enzymatic action, efflux pump systems, binding at peptides, and precipitation9.

Moreover, genes also play an important role in microbial nanoparticle biosynthesis. The resistance of microbial cells to metal ions during inward and outward transportation leads to precipitation. Accordingly, it becomes pertinent to understand the interaction of the metal ions with proteins, DNA, organelles, membranes, and their subsequent cellular uptake. The elucidation of the mechanism also allows us to control the shape, size, and monodispersity of the nanoparticles to develop large-scale production according to the required application9.

Furthermore, there are several aspects to consider while making GMOs. It is important to consider what kind of genes will be incorporated in these organisms, because this influences the precautions that have to be taken. Important questions can be: Will the GMO replicate itself easily? What would happen if the GMO escapes into the environment? Can your GMO produce dangerous compounds?

What we did

To work safely with GMOs we had an elaborate GMO safety training from our safety officer Marc Fluttert. This course taught and refreshed knowledge on how to properly work with GMOs. To keep this knowledge fresh throughout the project the Lab & Safety manager designed a Kahoot quiz for the team.

Lab safety when working with GMOs is crucial in any project. However, one should also take into account the possible effects of GMO contamination outside the lab environment. To address this issue, we worked together with the Dutch National Institute for Public Health and Environment (RIVM) to implement Safe-by-Design. RIVM developed this concept to help students assess the risks involved with their projects, even 'beyond the laboratory’.

Using a cell system or a cell-free system?

Now that you have obtained your GMO for optimal nanoparticle synthesis, you have to determine how to use this GMO in the production pipeline. Here, we discuss our choice of using a cell-free system in relation to safety.

Cell-free system

There are some examples of why you would want to make use of a cell-free system in an iGEM project, some recommendations from previous teams are:

  • No DNA mutation of your genetically engineered device because there is no DNA replication10.
  • No selective pressure on your genetically engineered device because the system is non-living and does not undergo natural selection10.
  • No self-replication of your genetically engineered device leads to a fixed amount of DNA being expressed and more control over the rate of expression10.

For our project , we used a cell-free system to ensure that our final product was free of GMOs. Alternatively a cell system can also be used for the biological production of nanoparticles. Read more on cell systems in the drop down below.

A big advantage of a production pipeline with a cell system is that it can, if provided by the necessary resources, keep a continuous nanoparticle production. However, proper purification of the nanoparticles from the cell system while making sure that there are no GMO contaminations can be a big challenge. Possible extraction methods will be discussed in depth below. However, these methods are not feasible in a completely closed system. This renders the possibility of no GMO contamination very slim.

A possible problem with a cell-free system could be GMO contamination of the extraction machines. In some cases, prevention of this risk completely is not possible at all, for example, for size exclusion chromatography (more on this below). Or the addition of strict medical regulations because the end product could have been in contact with GMOs.

Due to the problems that one could encounter when trying to extract the nanoparticles from a cell system we would recommend minimizing the use of a cell system in an iGEM nanoparticle project.

What we did

We aimed to use these biologically produced nanoparticles for medical purposes, thus it is vital that the final product is GMO free (See the interview with Aaike van Vught, CEO of Vsparticle). As such, we decided to use a cell free system elaborately described under our Project Description page. In addition to reducing the potential presence of GMOs in our final product, this system enables the use of a variety of extraction procedures, not available while working with GMOs.

Extraction

Before we can start the extraction of the synthesized nanoparticles, we have to make sure it is completely free of GMOs.

Sterilization

The sterilization processes of nanoparticles by autoclaving and filtration are two of the most utilized methods in the pharmaceutical industry but are not always viable options11. Autoclaving is highly discouraged, as the high temperatures of autoclaving could possibly change the morphology of the nanoparticles, causing a change in absorbance and thermal conductive potential. Highly effective and low-processing-time options for sterilizing nanoparticles for medical purposes are Gamma-radiation and UV-radiation11. Thus, we have detailed some options with the advantages and drawbacks in relation to safety:

In this method, a sample is spun at an extremely high G-force, which makes it possible to separate components of the sample in a gradient. With this method, it is also possible to separate nanoparticles by size. This can be done with a relatively simple sucrose gradient13 . See used protocol14.

The synthesized nanoparticles will not all be homogeneous, which is why this method can be extremely helpful. One can make a gradient and extract the different fractions. In this way, it is possible to extract nanoparticles of a certain desired size.

There are a few downsides to this method though: an expensive ultracentrifuge is required for this technique. Additionally, this methodology is only useful if you want to extract small amounts of samples since it would be inefficient to use it on a bigger scale.

Safety-wise this method is not ideal because ultracentrifugation of the sample will generate aerosols. To avoid contact with aerosols you need to wait at least an hour before it is possible to work with the samples.

SEC is a column liquid chromatographic technique commonly used for the separation of macromolecules in solution. Typically, SEC columns are packed with small, rigid porous particles of sizes ranging from 3 to 20 µm and pore sizes from 50 to 107 Å. SEC separates molecules according to their size in solution or, more specifically, their hydrodynamic volume. The larger molecules in a sample elute before the smaller molecules because larger molecules either enter fewer pores or sample a smaller pore volume of the column packing material (depending on whether the column is of a mixed-bed or individual pore size) than their smaller counterparts15.

This is a very promising technique to use for nanoparticle extraction because one can design columns specifically to suit the extraction of the desired nanoparticles. In literature, you can find a variety of eluents, columns and detection methods to suit one's needs.

There is a significant challenge in the SEC analysis of metal nanoparticles in measuring their adsorption to the column packing material. This means that part of the sample will remain inside the small rigid porous particles. Adsorption can cause several problems in the SEC analysis of nanoparticles15. Secondly, buying or constructing these columns can be an expensive task. And these expensive columns can only be reused for a limited amount of time, so keep this in mind when you choose this as a potential extraction method.

Field flow fractionation (FFF) is a conventional method that employs several external force fields. A field is considered effective if its strength and selectivity are sufficient to achieve separation. Typical fields include cross-flow streams, temperature gradients, electrical potential gradients, centrifugal force, dielectrophoretic force, and magnetic force12.

This can be a powerful technique to separate different types of nanoparticles. However, not every iGEM team will have access to such an expensive machine. So, this is not perse an optimal extraction method for iGEM nanoparticle synthesis.

What we did

We tried the ultracentrifugation extraction method. To keep the risk of aerosol exposure to a minimum, we waited an hour after the ultracentrifugation before opening the tubes. This made the time allocated for ultracentrifugation extraction almost twice as long. However, we highly advise other iGEM teams to do the same. Nanoparticle inhalation due to aerosols is one of the most dangerous ways to get exposed16. See the result section for the outcome of this extraction method.

Ethical concerns

Treating a patient with something potentially harmful

The administering of heavy metal nanoparticles can have negative side effects, this is due to some bio-reactive properties of metals.

However, these treatments are not intended to be used on healthy individuals - they are meant to treat a person who is already at a later stage of cancer. This treatment could potentially come with negative health effects, however, these side effects may be negligible in the face of a new innovative form of cancer treatment.

There is conflicting evidence on the general health effects of nanoparticles, let alone the effect of bimetallic urchin-like nanoparticles. However, at this time there is a huge amount of research being conducted17. Luckily, gold nanoparticles have been successfully used in other medical treatments, therefore we can expect more research in the coming decade.

Generating nanoparticle waste

The nanoparticles that are used in PTT will remain inside the patient for a certain amount of time. There are some studies done on the lifespan of a nanoparticle within the patient, but unfortunately not enough to make conclusive statements. However, we can assume that eventually, the patient will naturally dispose of these particles18.

In the hospital, this does not cause problems because hospitals have a specially designed closed sewage system. This closed sewage system makes sure that hospital sewage is separated from normal sewage and prevents direct contact of hospital sewage with the outside world. The hospital's waste is disposed of by a specialized company.

However, when the patients are discharged from the hospital, the nanoparticles could end up in the normal sewage system. The material in the sewage system could come into contact with surface water, potentially contaminating it.

The Dutch government strictly measures the number of nanoparticles in drinking water and in open waters. This is due to the large daily use of all kinds of nanoparticles in consumer products. Regular water treatment plants already filter out nanomaterials from the water19, it is therefore unlikely that the waste from PTT would cause any significant environmental impact.

GMO-produced medicine

The idea of injecting a patient with GMO-related material can be a controversial topic due to the limited understanding of the general public on GMOs. However, our nanoparticles are not GMOs themselves, rather we have only used GMOs in the production of our nanoparticles. Thanks to the use of our cell-free synthesis the final product is not a GMO just a product of it. The European Medicine Agency (EMA) does not enforce any extra GMO regulations on medicine that was produced with the help of GMOs, as long as the medicine itself is not a GMO. More information about our project and EMA can be found on the human practices page

Biosecurity and Dual-use

A possible dual-use application of our project could be the following:

A party with malignant intent could use our nanoparticle synthesis protocol to make nanoparticles that are harmful, very small silver nanoparticles for instance or maybe even small platinum nanoparticles. For more information about the possible effects of nanoparticles check out the Human Practices page.

Biosecurity & Dual-use:

Biosecurity: procedures or measures designed to protect the population against harmful biological or biochemical substances.

Dual-use: 'dual-use' implies that the biological agents and knowledge about these agents can be used for two different purposes. May it be for positive or negative goals.

RIVM Safe-by-Design

As part of biosafety and security, we worked together with the Dutch National Institute for Public Health and Environment (RIVM) to implement Safe-by-Design. The RIVM developed this concept to help students assess the risks involved with their projects, even 'beyond the laboratory’.

Thanks to talks with multiple experts in the field of safety we have integrated safety successfully throughout our project. For instance, we talked with the GMO safety officer of our institute and applied his advice by using a cell-free system approach. We have talked to lots of companies about the design of our final product and development. Besides companies, we contacted several doctors to ask for their professional opinion on the design of the project. We have developed an infographic to visualize the risks that could affect each of our stakeholders.

Infographic to visualize the risks that could affect each of our stakeholders.
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