Product Design

Framing the problem

Head and neck cancers account for approximately 900.000 cases and over 400.000 deaths annually on a global scale1. In the Netherlands, more than 3000 patients are diagnosed each year2. Head and neck cancer treatment can include surgery, radiation therapy, chemotherapy, immunotherapy, or a combination of treatments3. Unfortunately, these current treatments come with strong adverse effects. Patients suffering from head and neck cancers are therefore in strong need of a more specific cancer therapy with fewer side effects.

Photothermal therapy (PTT) could be the solution. PTT is a novel approach utilizing near-infrared (NIR) light absorbents capable of converting NIR light to heat. The produced heat ablates cancerous cells16,17. As PTT is minimally invasive, it is currently receiving increasing attention as a potentially effective therapeutic treatment.

As detailed in our project description, metallic nanoparticles are ideal absorbents for use in PTT. However, formerly produced nanoparticles have a suboptimal size and shape for the therapy. Additionally, the creation of nanoparticles as accomplished by chemical and physical methods leads to the presence of some toxic chemicals absorbed on the surface that may have adverse effects in applications.

In a comparative study by Pandit et al.18, biologically produced nanoparticles were found to have considerably lower cytotoxicity than wet chemical nanoparticles, proving their safety and ability to be used widely in biomedical applications. Compared to physical and chemical approaches, bimetallic nanoparticles that are biologically produced also show great activity in biomedical applications19. Combined with the relatively low production costs, this makes them interesting for PTT.

Product design

In order to design our optimal product, we spoke to lots of stakeholders and tried to take into account each of their opinions. Read more about the interviews with the stakeholders and our subsequently taken steps here.

Additionally, we did a lot of literature research on how to design the perfect nanoparticles for PTT. The ideal PTT candidate should have the following characteristics: 20

  • Suitable size and of uniform shape.
  • Good dispersibility in aqueous solutions. This is necessary for intravenous application.
  • Convert light in the near-infrared region (NIR) to heat.
  • Sufficiently photostable. This is to ensure enough diffusion time to reach tumors before losing their photosensitivity.
  • Low or no cytotoxicity in living systems.

Near-infrared region (NIR)
Wavelengths measuring from 750 nm to 1,300 nm are generally considered to fall into the near-infrared region. For application in PTT, the NIR light needs to penetrate tissue to be able to reach a tumor. Light between 750 and 900 nm has maximal penetration depth in the tissue21. Therefore, nanoparticles used in PTT need to have an optimal absorbance and heat conversion in this specific region. After activation with an infrared laser, the heat will kill the tumor cells.

The composition of materials, shape, and size of these nanoparticles determine the necessary characteristics mentioned above. With help of previous studies and our integrated human practices, we were able to optimize the protocol for synthesis of the nanoparticles and develop a prototype.

As explained in the dropdown above, we found that the optimal nanoparticle design consists of an urchin-like nanoparticle, with a silver core and golden spikes. The most suitable size lies between 20 and 150 nm, and within this range, certain sizes are more favorable for specific aspects.

After a few hard weeks in the lab, we were happy to have promising results. Fortunately, our produced nanoparticles satisfied multiple of the desired properties for PTT, since they showed absorbance at 800 nm and had an urchin-like shape. The size fell in the right range of 20-150 nm, as can be seen in the results section. More importantly, the heat experiment revealed that the nanoparticles were able to convert the light of a NIR laser into heat with a ΔTemp of 7.1°C and a PTT conversion efficiency of 44.3%.

However, our prototype needs some adjustments in the future to optimize its suitability for PTT. You can read more about our future vision and challenges that come along later on this page. But first, let’s talk about how our nanoparticles will be implemented and impact the world.

Implementation

Implementation plan

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Fig. 1 | The seven plans of our implementation plan.

In our implementation plan, we focused on seven different steps that are key to a successful implementation, as shown in Fig. 1. It represents the journey of our nanoparticles from the iGEM project to the product for photothermal therapy. In these steps, we included not only the practical considerations for a successful business but also the safety aspects for employees, healthcare professionals, and of course: the patients.

During our interview with prof. Henk Noorman from DSM and TU Delft, we learned a lot about upscaling. We discussed every step in the production process and how to perform this certain step on a larger scale. Since we have quite a specific purpose for production, we would be able to produce on a relatively small scale. Therefore, Prof. Noorman advises us to focus on quality instead of speed, as poor quality requires rework and time gained will be lost again.

Even though upscaling is very important for the implementation of Binanox’s nanoparticles, it is a step that needs to be executed only once in a long period. For this reason, the upscaling is done only once, while the other steps in the implementation plan are run through every batch of production.

According to prof. Noorman, we could do the first upscaling in the lab, which is inexpensive and relatively quick. So, first of all, in order to prepare for work on a larger scale, we need to have a lab space in which we are allowed to operate bioreactors. BioPartner, located at the BioScience Park, seems to be capable of offering this. BioPartner is one such group of companies that can offer laboratory space and comply with all the regulations of running a lab safely.

After finding a place to work, we can start our actual production process. The first step of this process is to transform Escherichia coli (E. coli). This transformation can be done on a regular lab scale. The natural competency of E. coli is very low or even nonexistent, which is why the cells need to be made competent for transformation, for example by heat shock or by electroporation. Premade competent cells also exist, for consistency and to save time. These competent cells have a suitable transformation efficiency and minimize batch-to-batch variability. With these competent cells, the transformation can be done. Negative and positive controls should be included in the transformation step to evaluate the success of the experimental procedure. Subsequently, after growing in a medium, the cells are plated on LB agar with agents for identification and recovery of successful transformants.

The next step in our production process is cultivating a culture of transformants. For this, we will naturally need bioreactors. Prof. Noorman advises us to first use a bioreactor of 1000 L, with which he roughly estimated to produce about a kilogram of nanoparticles per batch. According to estimations based on our proof of concept, about 19 mg nanoparticles would be required to kill an average-size head and neck tumor. Since synthesis takes about 24 hours, approximately 52,000 treatments can be made in one day. Taking a time margin into account, it would be possible to produce about 100 batches a year, making 5,200,000 treatments yearly, which is about 55% of the world's incidence.

According to Dr. Noorman, working with E. coli should not give many complications, since it is often worked with on a large scale. There should be accounted for loss of yield when scaling up, since the conditions in a bigger bioreactor may be less optimal for the micro-organisms40. Therefore, there is a smaller risk of losing relative yield by scaling up to 1,000 L than to 10,000 L. To be able to know more about the scalability of our nanoparticle production, we should do experiments using 10 L. According to Dr. Noorman, production on this scale is quite representative of that of a 1000 L. The relative yield will be about ten times lower in a bioreactor compared to a small scale. After the scalability experiments, we would need to make only slight adjustments, i.e. the amount of oxygen or the composition of the medium, to assure our production process is correct.

Dr. Noorman also mentioned the plastic single-use bioreactors from Sartorius or Applikon, which are delivered sterile and can be modulated in size. Additionally, a permanent bioreactor poses a risk of a wrong purchase concerning the size, when no experiments have been executed beforehand in a plastic bioreactor. Thus, a plastic bioreactor would be ideal in our case since we can then adjust our production based on the demand. Our approach will be to first get one bioreactor of 1000 L and then go for a permanent and sustainable bioreactor.

In order to get the content of the cells out, we should do an autolyze. On a large scale, this can be done by various enzymes that encourage the breakdown of the cells. This could be done in the same bioreactor or in a different one.

After the autolysis, the supernatant should be collected. We would need to get an industrial sediment centrifuge that allows for separating bigger volumes.

Subsequently, to remove any contamination the volume needs to be filtered. This can be done with an industrial filter press. Specifically, the filter press separates the liquids and solids using pressure filtration, wherein the liquid is pumped into the filter press and is dewatered under pressure. A suitable filter size would be 200 nm, just as we did in our experiments.

Then, gold and silver should be added. To make the reduction of nanoparticles possible in a cell-free extract, we should ensure that a redox reaction can take place. For this, a cofactor regeneration is necessary41. According to Dr. Noorman, a suitable redox enzyme would be 2-propanol. Dr. Noorman brought to our attention that high chloride concentrations, which may arise due to the presence of HAuCl4, could negatively impact bacterial cells. However, we have alleviated this problem, and subsequently the cost of solving it by using a cell-free system, as detailed above.

After the formation of nanoparticles in each batch, we should analyze these. This can be done with the same methods and by taking samples out of the batch. These methods include the TEM, the spectrophotometer, the zeta sizer, and the heat conversion experiments.

Whenever our nanoparticles satisfy the desired properties, it is time to isolate them (as detailed below in the isolation dropdown). Last but not least, the nanoparticles should be targeted with antibodies specifically for head and neck cancers. In order to make this possible, we should make sure that the exterior of the particles contains active groups. For this, we could use a coating, rich in ions. An antibody must have a ligand so that there arises a ligand/tail affinity, leading to the attachment. According to Dr. Noorman, this can be easily glued together via an affinity solution. In order to do this on a big scale, several companies, like Eurogentech and Genscript offer custom monoclonal and polyclonal full-length antibodies for biotech and pharmaceutical companies.

In order to enforce worker safety, clear protocols for employees should be made, and whenever possible closed systems should be used. Also, employees should wear protective clothes like a mask, helmet, jacket, and steel-toe shoes. As little manual work as possible should be done and employee training should be provided. Furthermore, there should always be supervision, whereas there is also external supervision. RIVM, the Dutch National Institute for Public Health and the Environment has outsourced the supervision to COGEM, the Commission of Genetic Modification. This Commission is moreover competent in giving licenses for working with GMOs.

Generally, it is economically more advantageous to carry out production in high-resource areas. However, according to prof. Noorman, this will not have such tremendous effects on our production process, since we produce on a relatively small scale. Hereby, we won’t need large volumes of raw materials, which means that it is not necessary to establish in countries with for example high resources in gold. Therefore, prof. Noorman advises us to establish somewhere where working safely and cleanly is the norm and where high technology is available. This indicates that working in prosperous countries is most interesting for Binanox, where it is also easier to cooperate with hospitals.

Also, we consider an environmentally friendly process extremely important. This is why transport also plays an important role in the choice of location. Since we have chosen to aim our SOM at the Dutch market for head and neck cancers, we will have to transport our product to the Netherlands. Therefore, it is logical to establish our company, including our production process in the Netherlands. More specifically, this would be ideal in Leiden, where our team is already located. The BioScience Park in Leiden has all the technology, knowledge, and innovation we could take advantage of.

When considering environmental effects, an important factor is to have closed a sewage system during production. This prevents contamination and release of nanoparticles into the environment. The wastewater should be purified by a water treatment plant that you can buy ready-made nowadays. In order to filter out the metal nanoparticles, we can use biohydrometallurgy. This method uses biological agents to isolate valuable or harmful metal ions from water. By the time we will need this method, we could contact prof. Mark van Loosdrecht from the technical university in Delft (TU Delft), who is an expert in this area. After purification of the water, this can be discharged after testing. External companies are called in for waste processing, whereby any left intact DNA is degraded.

During our production process, we will of course follow the Good Manufacturing Practices, the quality assurance system for i.a. the pharmaceutical industry, with its five components shown in Fig. 2.

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Fig. 2 | The 5 P's of Good Manufacturing Practices (GMP), picture adapted from C. Sheen Escano53.

Naturally, it is very important to isolate our nanoparticles from any cell debris or reducing agents whose effects on the human body are known or unknown. Since these agents are not necessary for PTT to work, they can be removed without influencing the treatment. This must be done in order to avoid any possible adverse effects during the therapy. To do this on a large scale, we will need a specific method, which we discussed with Dr. Henk Noorman from DSM.
Our nanoparticles are heavier than the organic fraction, which indicates that this can be done with a general centrifuge. Then the nanoparticles should be washed, after which they should be centrifuged again. In order to make this work, the density of our nanoparticles should be known. If the density is higher than 1.1 kg/L the method mentioned before should work. However, if the density is lower than 1kg/L then we should use a nano-centrifuge. After isolation, the nanoparticles should be mixed with a liquid, in order to make them easier to transport and safer to work with.

After our nanoparticles are produced and isolated, they should be validated. This will result in a slight delay in the process from production to end-user. However, the validation can be done by taking small samples for larger batches, so the delay will be relatively small. Moreover, it is crucial to ensure the quality of our nanoparticles. In order to gain the trust of organizations like RIVM and the World Health Organization, we should draw up requirements of characteristics, such as sufficient homogeneity or only a certain allowed deviation of the length of the spikes, which we can check with our validation techniques, which are described earlier. This will all be properly documented as the Good Manufacturing Practice (GMP) suggests. Recommendations by large organizations like these will increase the trust of doctors, hospitals, insurance companies, and last but not least, the end-user.

Since we chose to aim for the Dutch market in the short term and since our production process takes place in the Netherlands as well, the distance of transportation and thereby the impact of this is relatively small. Before entering the Dutch market, we should get approval from the European Medicines Agency.

However, in the medium-long term, we are planning to cover Northern American and the entire European market as well, since these score the best in size, potential, and access. In order to make this happen, we should get additional approval from the Food and Drug Administration (U.S.) and The Canadian Agency for Drugs & Technologies in Health (Canada). We aim to take all these agencies' regulations and demands into consideration during our clinical trials so that we have a high chance of getting approval without having to spend unforeseen additional costs afterward. In the long term, we even plan to aim for providing Binanox’s treatment all over the world, which you can read about here.

During transportation, it is crucial that the nanoparticles keep their characteristics making them suitable for PTT. Therefore, validation of the nanoparticles after transportation should be done until repeating satisfying results occur. Transportation will be done in plastic barrels, steel drums, or plastic tubes, depending on the volume needed. To add extra protection against spillages and contamination, metal and plastic liners can be inserted.

Finally, our product will reach the End user, the patient, whereby the product will be used in the therapy against head and neck cancers. The nanoparticles are delivered in a liquid, proper dosage would be dependent on the weight of the patient and size of the tumor. Binanox could provide doctors with training on how to utilize our nanoparticles during therapy and how to maintain safety for both patients and hospital employees at all times.

Sustainability

Binanox considers sustainability as a crucial aspect of the entire implementation plan. With the action written down below, we will contribute to these certain Sustainable Development Goals (Fig. 3):43

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Fig. 3 | Sustainable Development Goal 3 and 12, as set up by the United Nations43.

Green energy
Green energy often comes from renewable energy technologies such as solar energy, wind power, geothermal energy, biomass, and hydroelectric power. However, in order to be called green energy, the energy resource cannot produce pollution, such as with fossil fuels. This means that not all sources used by the renewable energy industry are green. For example, power generation that burns organic material from sustainable forests may be renewable, but it is not necessarily green, because of the CO2 produced by the burning process.

First of all, we aim to use green energy for all our processes. Ideally, 100% of our energy would come from our own solar panels. Secondly, we only want to use products that have a verified sustainable production process. For example, we would prefer to buy raw materials from verified sustainable sources. If this is not possible, we aim to have transparent discussions with possible suppliers about responsible production, to find the most sustainable products.

It is also important to keep the organic production process going in order to lose as little energy and materials as possible. Therefore it is important to add 2-propanol as discussed in the upscaling, which should be produced in a sustainable way just as well, for example as discussed by Panjapakkul and El-Halwagi44.

Furthermore, keeping our waste production and disposal in check is crucial. Pharmaceuticals are widely distributed and there is a consistent global increase, corresponding to the increase in the generation of pharmaceutical waste. During the manufacture and use of pharmaceuticals, lots of materials become contaminated with the active pharmaceutical ingredient, in our case the nanoparticles, increasing the waste volume45. The waste disposal of the nanoparticles has been detailed in the implementation plan and Safety page.

In order to prevent lots of waste during our implementation plan, we try to stay away from unsustainable materials, like the plastic barrels used while transporting and the plastic bioreactors used during our production process, after we have executed our upscaling experiments in the plastic single-use bioreactors. Even though cleaning and sterilizing steel barrels and bioreactors can bring more costs and time use, we consider sustainability more important than making the most profit. Also, we will try different methods of increasing the preservability and stability of our nanoparticles. As mentioned in our implementation plan, in an ideal situation, our nanoparticles can be stored for a longer period of time. This leads to less waste of medicines and less unnecessary transport.

Moreover, pharmaceuticals are often excreted unchanged and can reach the environment46. Therefore, we will install a closed sewage system and a water treatment plant to prevent contamination and the release of nanoparticles into the environment. In order to filter out the metal nanoparticles, we could use the method of biohydrometallurgy, as described before. Also, research should be conducted about the period of time in which the nanoparticles might be excreted by the body. Then, measures should be taken to prevent these nanoparticles from being excreted into the open sewerage. An example could be to keep the patient hospitalized for a certain amount of time, whereby the hospital has closed sewerage.

Impact

Making an impact analysis is an important part of the project. The direct impact of our product would be to improve the lives of patients with head and neck cancers. Assuming that our product will successfully run through the clinical trials, our therapy will lead to a shift in demand from chemo-, radio- and immunotherapy to PTT with Binanox’s nanoparticles. In the long term, this will lead to significant growth in the Dutch economy. Also, since PTT is less invasive than currently used treatments, patients will need fewer hospital visits and thereby less transportation.

The indirect impact would be the emissions produced by the production, transportation, and disposal of the product. Also, the long-term effects of getting in contact with the nanoparticles, during production, transport, therapy, or via the environment, are indirect impacts of our product. Only little is known about the effect of nanoparticles on healthy individuals. However, we aim to reduce the release of nanoparticles into the environment fully, as described before.

Challenges & future vision:

After completing the synthesis of the nanoparticles, we expect to come across several challenges that need further research and to be accounted for.

Efficacy

Different methods for the localization of the nanoparticles to the tumor need to be explored. Some known strategies are intra-tumor injection, active targeting, biometric targeting, and programmed targeting47. A previous study with nanoparticles coupled to an antibody targeting the epidermal growth factor (EGFR) used in PTT has shown promising results48. Ideally, we would use antibodies specific for head and neck cancers, to target these cells even more specifically.

Another challenge for the efficacy to work might be the low preservability or stability of the nanoparticles. Different packaging materials and additional preservatives should be tested for this. This is very important, since preservability is important for our sustainability goals, as described earlier.

Safety

One of the most important aspects of a new medicine is its safety. PTT is promised to be a minimally invasive treatment. However, unexpected toxicity should be accounted for. In order to lower possible cytotoxicity, a coating could be used. Tests on models with and without different coatings should be compared on efficacy and side effects. Polyethylene glycol is a coating with high potential to use successfully and safely on golden nanoparticles for drug purposes49.

Over and above that, the distribution through the body and the natural clearance of the nanoparticles should be checked in animal models. The cytotoxicity could be tested in both animal models and human organoids before entering clinical trials50. After finalizing the preclinical experiments, the newly developed therapy for PTT should be tested in the clinical trials, including additionally needed devices, e.g. NIR lasers. In the course of these trials, efficacy and adverse effects will be tested on test patients.

Market expansion

After Binanox’s treatment has been on the market in Europe and Northern America, we aim to offer our treatment all over the world, in line with human rights. Even though in reality at least half of the world's population cannot obtain essential health services51, the Universal Declaration of Human Rights states that every human being has the right to health care.

Whenever it would have been a possibility, we would have aimed for this at the very beginning of our market entry. However, in reality, this is hard to acquire in the short to medium-long term. Since we consider the quality of our product very important, we prefer to focus on production on a smaller scale first. Moreover, costs before market entry are very high and therefore high revenues must be made quickly after market entry. This is not achievable whenever we aim for countries that have a low number of cases, low purchasing power, or accessibility.

In order to make our product accessible in low-income countries as well, we should tailor the price of our treatment to a country's capacity to afford these. In order to make this happen, we could let high-income countries pay a higher price of which a part will fund the purchase of treatments in lower-income countries. Since we will try to cover the market worldwide when Binanox already has existed for a few years, we assume we will then make high profits. Another option, therefore, is to use part of our profits to invest ourselves in lowering the prices in low-income countries.

Once Binanox is a well-established company, research on other applications could be conducted. Nanoparticles have various applications, as shown in Fig. 4. For example, metal nanoparticles have a large potential in the aerospace industry52. However, non-complicated nanoparticles can be synthesized chemically, which is generally cheaper. Thus, only whenever there is a value proposition to the synthesis for this new application, further actions should be taken.

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Fig. 4 | Different applications of nanomaterials, image inspired by A. Aslam et al.54

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