Prof. Dr. Dick Sterenborg is an expert in phototherapy photodynamic therapy (PDT) and currently works at the Netherlands Cancer Institute and Academic Medical Center in Amsterdam as a professor in biomedical engineering and physics. Since there are currently very few experts in PTT worldwide, we aimed to learn more about the therapy by investigating associated therapies like PDT. Further, he enlightened us on what considerations we should make when working with a NIR laser for treatment purposes and gave us some ideas for a proof-of-concept.

Integration

Prof. Dr. Sterenborg advised us to produce nanoparticles with an absorbance between 700-900 nm because this wavelength causes the least damage to surrounding tissue, therefore ensuring safety. This indicates that absorbance will be one of our most important measurements, and based on that we should use our model to find the optimal parameter to produce the optimal nanoparticles for PTT.

Further, we have decided to have our proposed Implementation to be a group of cancers that are relatively close to the skin, namely head and neck cancers. This is due to the limited penetration of a NIR laser as Prof. Dr. Sterenborg informed us.

As for the Proof of Concept experiments, we have tested whether our nanoparticles can convert the excitation by the NIR laser into a temperature increase. Due to the resources available to us, we have slightly altered our experimental set-up compared to how Prof. Dr. Sterenborg advised us. Instead of using a thermal camera, we measured temperature increase in media with the use of a temperature probe.

Furthermore, it will be important to test the stability (see our interview with Dr. Aquilles Carattino) of our nanoparticles when heated, since Prof. Dr. Sterenborg informed us of a study which showed the instability of gold nanorods when heated. Instability of our nanoparticles could indicate decreased effectiveness, however, on the other hand also potential reduced toxicity to the body and environment.

The interview

Prof. Dr. Sterenborg re-emphasized at the start of the interview the differences between PDT and PTT. This is very important to keep in mind since we will be using some of the information on PDT to learn more about PTT, but results found in studies using PDT are not completely generalizable to PTT.

However, the most valuable part of our discussion was learning more about why progress in the development of therapies like PDT and PTT is extremely challenging. Regulatory authorities like the FDA often require products that combine a pharmaceutical and a device to be evaluated provided together. In other words, when someone is aiming to produce a new therapeutic as we do, we will have to provide both the pharmaceutical (the nanoparticles) and the appliance (the laser). Most large pharmaceutical companies avoid the complication of adding technical devices like lasers and most tech companies with the ability to produce a high-quality laser, cannot produce a pharmaceutical. This type of therapy is located at a cross-section of development and is therefore often overlooked. Furthermore, it is extra difficult for start-ups in this area to break through due to the extra boundaries set having to deal with both an appliance and pharmaceutical and needing expertise from three different areas: oncology, pharmaceuticals, and hardware developer. The progression is further hampered by the prospect that the many costs going into navigating the regulations will not be compensated enough in the improvement of the current therapies for some cancer types. So, even though there is a lot of free space for science to develop in this area, there is no easy way of transforming this science into a usable application and that causes the area to slowly die out.

Prof. Dr. Sterenborg was also able to enlighten us more about the practicalities of using laser-mediated therapy. He advised us on the wavelengths we should aim at with our particles to not cause harm to the surrounding tissue, namely 700-900 nm. He also gave us the hint to look at stained glass. In red stained glass, gold nanoparticles at sizes 30-50 nm are used. Of course, we would need slightly larger nanoparticles since they are in tissue.

Further, he informed us of a study done about a decade ago in Twente, where they found that gold nanorods will revert to a sphere when excited by a laser due to conformation stability. This means that we will have to test and investigate if and how our nanoparticles will alter shape when excited.

Finally, Prof. Dr. Sterenborg has some suggestions for our experiments and proof of concept. One of the suggestions he made to test whether our nanoparticles will be able to convert the energy from the laser into heat, is to use cell culture and then measure the increase in heat using a thermal camera.

Supplementary literature

Esmail et al.4 showed earlier this year that silver nanoparticles produced using the bacteria obtained from the Zarshouran gold mine have an absorbance between 400-470 nm with a size distribution of 20-70 nm. Similarly, Zaki et al.⁵ found an absorption between 350-460 nm for nanoparticles with a size distribution of 15-50 nm. Funnily enough, Cionti et al.⁶ showed that silver nanoparticles in stained glass create a yellow color, from this, we have derived that the excitation of silver nanoparticles is a bit lower than that of gold nanoparticles.

For gold, the absorbance spectra are between 520 and 530 nm for average nanoparticles diameter between 12-41 nm,⁷ with a shift to red absorbance as the diameter increases. Luckily, we were aiming to obtain nanoparticles with a diameter between 50-100 nm, meaning that absorbances with solely gold were slightly higher but still under 600 nm. We were able to shift the absorbance more towards 750-900 nm with the help of changing morphology⁸. Furthermore, nanoparticles with 6 spikes show increased tumor uptake⁹.

Joan Birkhoff is PDT coordinator at the Antoni van Leeuwenhoek hospital in Amsterdam. We had several meetings with Ms. Birkhoff while integrating feedback from other interviews and team meetings. Ms. Birkhoff provided insight into how PDT (photodynamic therapy or light therapy) is applied to patients and the disadvantages and advantages of this form of therapy. After talking to RIVM, we further interviewed Ms. Birkhoff about PDT waste management.

Integration

Ms. Birkhoff informed us that the photosensitizer for PDT is delivered in falcons to the hospital. If we do the same with our nanoparticles (which is the most logical) we are making a trade-off. On the one hand, it means we are better able to protect the hospital staff, while on the other hand, we have to put more work into investigating shelf-life. As such, we investigated what happens when we limit clumping over time by adding surfactants, see notebook page surfactant experiments, as well as how our nanoparticles would behave over time without adding surfactants. The latter was investigated by obtaining TEM pictures after leaving the nanoparticles to rest in medium for approximately 2 weeks.

Ms. Birkhoff explained that PDT results in light sensitivity (i.e. burn wounds if exposed too long) and we can expect that PTT has similar drawbacks. This is largely because wavelengths emitted by the sun partially overlap with wavelengths from the NIR spectrum. However, to limit the side effects experienced, we have investigated targeting methods like using antibodies (see our interview with Mr. Dimitri Pappaioannou). This suggests light sensitivity only in the target region as opposed to the whole body, limiting the risk of getting burn wounds.

These conversations with Ms. Birkhoff together with literature research stimulated us to investigate the option of taking head and neck cancer as the main proposed implementation, see Implementation. However, as Ms. Birkhoff explained, PDT is quite expensive. Therefore it motivated us to research more on regulations regarding coverage of treatments and affordability thereof, to investigate how PTT could be made affordable (for more information see the interview with Anke Hövels).

Interview 1

Ms. Birkhoff explained the current practice of using PDT. Currently, it is legally defined as a palliative treatment because of its high cost (it is labeled as an orphan drug), of advanced squamous cell carcinoma in the head and neck area, if previous therapy was not sufficiently effective and radiotherapy, surgery or systemic chemotherapy are not eligible. However, it is often used as treatment in combination with or without surgery. We were further informed about the steps taken when PDT is prescribed. For example, in nasopharyngeal carcinoma, surgery is performed first. Then, 6 weeks are waited as treatment does not work on bleeding tissue, after which PDT is applied. Prior to PDT, the patient is given an injection of the photosensitizers Foscan. This undiluted liquid is administered intravenously 4 days before the laser treatment to allow the liquid to infiltrate the tumor. The tumor is then treated day 5 with the 652 nm laser, after which the patient can go home and recover. The patient does receive a lux meter to take home before treatment to measure light exposure. This is due to light sensitivity during the first 2 weeks. This light sensitivity is caused by Foscan. However, a build-up schedule with regard to light gradually reduces light sensitivity.

Compared to surgery, PDT has some benefits. The major benefit is that PDT causes a type of burn wound, which stimulates bottom-up healing. In other words, PDT preserves the ability to form new tissue, surgery removes the tissue completely.

Compared to surgery, PDT has some advantages. The main advantage is that, on average, PDT gives a beautiful healing, for example, when a lip is treated. Anesthesia is short during treatment because the whole treatment with the laser lasts only 200 seconds.

At Antoni van Leeuwenhoek Hospital, PDT therapy is used for head and neck tumors. Photodynamic therapy is also used for skin cancer with prior local application in the form of a cream.

Finally, Ms. Birkhoff outlined how many people per year receive PDT for conditions in the head and neck area. An estimated 20-30 patients a year receive PDT at Antoni van Leeuwenhoek Hospital. Compared to the number of patients diagnosed with head and neck cancer annually in the Netherlands, 3174 diagnoses in 2021,¹³ this is very few. This is partly explained by the fact that there is only a limited indication for this treatment, namely the size, depth and location of the abnormality.

Interview 2

Based on the interview with RIVM and conversations within the team, we had some questions that Ms. Birkhoff could help with. The questions related to the disposal of treatment waste, how PDT is delivered to the hospital and how PDT is paid for.

We asked whether the injection fluid used in PDT is compounded at the hospital or in advance. This seems like a simple question, but it has important implications for the safety of hospital staff and what measures we should take to extend shelf life if necessary. However, for treatment PDT, Ms. Birkhoff was able to inform us that the injection fluid is supplied in vials.

An additional question concerns the disposal of treatment waste. In the case of PDT, these products are obviously contaminated with a photo-sensitive fluid. However, in the case of PDT, the waste will be contaminated with the metals gold and silver, which may have environmental implications, as discussed in the section on RIVM. In the case of PDT, the waste will be disposed of with general hospital waste.

Supplementary literature

To improve the shelf-life of nanoparticles we looked into surfactants. Surfactants have a multitude of roles when added during nanoparticle synthesis. One of these roles is to act as a capping agent¹⁴, limiting the aggregation of nanoparticles when left in solution and therefore aiding in prolonging shelf-life. Furthermore, surfactants have the ability to direct the morphology of nanoparticles^14,15. This is a very interesting aspect of surfactants for us and could be studied in the future.

In the case of our nanoparticles, our treatment-associated waste will be disposed of according to cytostatic and cytotoxic medicine ¹⁶ since there are no legal guidelines set for the disposal of nanomaterials¹⁷. This means it will be disposed of with other hospital waste and thermally treated. In the Netherlands, most hospital waste is disposed of by Zavin, who incinerates the waste by heating it to approximately 1000 °C¹⁸. This incineration will likely cause our nanoparticles to form bottom ash^17,19. This form of disposal seems to be highly effective at preventing emissions to the environment²⁰.

Dimitri Pappaioannou is director of business development at Oncolines. Oncolines is a Dutch based precision medicine services company in oncology and cancer immunotherapy. They use a broad panel of cancer cell lines to perform drug activity analysis. They have experience with working with clinical and preclinical biopharma companies and a wide variety of drug candidate type molecules including antibodies. We have been contemplating conjugating our nanoparticles to antibodies as a form of targeting.

Integration

Mr. Pappaionnou emphasized to focus our implementation on cancer that is close to the skin, with the addition that it should be accessible by blood. This last point is to ensure that our nanoparticles are able to infiltrate the tumor. To build up on the Implementation, we discussed with Mr. Papaioannou the importance of directed therapy and the options to bind our nanoparticles to antibodies. Mr. Pappaioannou advised us to look into linkers that could bind our nanoparticles to antibodies since it could give advantages like the aid in maintaining the desired surface plasmon resonance (the electron phenomena that enables our nanoparticles to convert the energy from the laser into heat) or giving the ability to separate from the antibody. After some literature review and a different interview with Dr. Djanashvili we saw that there is no specific need for either of the two, so there is explicit need for a linker.

It is to note that currently produced antibodies are not 100% specific as Mr. Pappaioannou explained to us. However, as our nanoparticles will only be a functional therapy in the future, we expect that antibody production will have been improved. Therefore we expect to be able to develop more specific tumor targeting with our nanoparticles.

Interview

Mr. Pappaioannou informed us that one very important consideration for the targeting of the treatment is, to ensure that the antibody that we use is specific to a protein expressed by the tumor we want to target. The use of antibodies is not 100% foolproof. Even if you can target the nanoparticles to be tumor-specific, there is still a chance that not every tumor cell will be targeted by the antibodies, meaning that some cells will escape.

Another point to test is whether our nanoparticles will be able to bind to antibodies. There are two options for binding our nanoparticles to an antibody. For the first one, we could bind our nanoparticle to the antibody and when the antibody binds to the cell, it is activated. In this case the design of a linker could affect the activity of our nanoparticles. The other option is to use the antibody to internalize our nanoparticles into cells, in that case it could be useful if the linker dissociates and releases the nanoparticle from the antibody.

Inquiring more about cell lines, Mr. Pappaioannou informed us that if we were to do cell line experiments, we should use a cell line that represents the tumor of our implementation and that expresses a specific target we can use for antibody recognition. Furthermore, it would be advised to test on multiple cell lines and use cell lines that we are able to use in downstream processes. Cell lines from the same tumor could express the target in equal amounts, but have different responses to the drug, in our case nanoparticles. Therefore, it is important that we test multiple cell lines from both the same tumor and different tumors.

One aspect that especially requires attention is what will happen to the nanoparticles after the therapy. How will they leave the body?

Going more into specificity, he advised us to look into antibodies that are already used and investigated for the target cancers. Furthermore, Mr. Pappaioannou mentioned that the delivery method is of importance as well. Again, he advised us to read into what is already being investigated or available on the market and build up on that.

Additionally, when inquiring about advice for a proof-of-concept Mr. Pappaioannou proposed to see whether our nanoparticles conjugated to antibodies will kill cancer cells while not harming healthy cells. This would mean that we have a very specific therapy. An essential part of any newly developed therapy is that it is better than any before in aspects like being safer, high efficacy, etc.

Lastly, Mr. Pappaioannou had some input on our potential implementation. He advised us to focus on cancer that is close to the skin and accessible by the bloodstream. When we mentioned the potential of head and neck cancers, he could see the potential it has since it is such an essential region for normal functioning, like swallowing and speaking.

Supplementary literature

Previous research has shown that it is possible to form antibody conjugated nanoparticles ²¹ and also for metallic nanoparticles²². This is due to the fact that proteins conjugate to silver and gold spontaneously²³. There have even been some clinical trials using antibody-conjugated nanoparticles²².

Integration

By inquiring through the contact form from the European Medicines Agency (EMA), we learned that we do not need to alter our plans significantly to be able to bring our nanoparticles to market. Furthermore, our nanoparticles do comply with the medicinal product definition for medical products given by the EMA. We do, however, need to have contact with Rijksinstituut voor Volksgezondheid en Milieu (RIVM, National Institute for Public Health and the Environment) to learn more about the possibilities for production using Genetically modified organisms (GMOs) and the legislation in the Netherlands.

Contact

We contacted the EMA for more information through the contact sheet on their website and got an answer to our questions. Based on this we gathered some information. First of all, our product would not qualify as a Genetically modified organism (GMO) since no GMO will be present in the actual treatment. We would, however, have to prove that our nanoparticles are a medical product. The definition given about this by the EMA is as follows:

Medicinal product = “A substance or combination of substances that is intended to treat, prevent or diagnose a disease, or to restore, correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action” ²³.

As previously mentioned, the final product is our nanoparticles with no GMOs, meaning our product will be legally discerned as similar to GMO-produced recombinant proteins (for example human insulin).

The last point we obtained information on is regulations regarding the production process. For this, the production plan will have to get approval and obtain environmental authorizations from the national authorities, in our case RIVM.

RIVM is (Rijksinstituut voor Volksgezondheid en Millieu) a governmental research institute in the Netherlands regarding population health and a safe living environment. It is the research institute that informs the ministry of health, welfare, and sport. We had two interviews with experts, Dr. Cécile van der Vlugt-Bergmans and Mr. Wessel Teunisse from RIVM. The first one was to give an idea of our project and to obtain advice from the RIVM representatives. The second interview was to update our progress and to ask some remaining questions.

Integration

Since our project is focusing on a biomanufacturing process, RIVM recommended that we investigate the production process more closely. This led us to interview Prof. Henk Noorman about the scale-up of production and worker protection.

Furthermore, the conversations with RIVM inspired us to investigate treatment-associated waste disposal for PTT with our nanoparticles and whether it would require additional precautions taken during product design by inquiring Ms. Joan Birkhof about the disposal of photosensitizers. Currently, nanoparticles can be disposed of safely using chemical waste disposal methods, however there is ongoing research whether this method is sufficient enough.

Lastly, we were given study material on safe-by-design, see Safety page, which we used to inform our experimental design. This, for example, convinced us to set up a cell-free system production process, which ensures biosafety and alleviates the necessity to take extra precautions for Genetically modified organism (GMO) waste disposal. Therefore, we experimented with this during our experiments.

Interview 1

During the first interview, we spoke a bit about our experimental setups. We informed Cécile and Wessel that we already have taken some precautions, like consulting the safety managers of the Institute of Biology Leiden at Leiden University. Furthermore, we were already considering the idea to produce our nanoparticles with a cell-free system, i.e., obtaining the proteins necessary and then adding the metal solutions to start the production of nanoparticles. This would limit the risk of leaking GMOs into the environment. On this topic, we were also given information and material for discussion to speak about safety-by-design and safety in biotechnology.

Additionally, Cécile and Wessel gave us inspiration for research topics. One aspect they recommended for us to look into is the production process. In our project, we aim to produce nanoparticles. However, we need to be able to translate our experiments in the lab into a functioning production process. This production process will warrant safety measures to be put into place. In line with this, Cécile and Wessel advised to consider safety protocols for potential future employees.

Encapsulation and potential toxicities are something we also had to look at. Encapsulation will limit the formation of protein coronae around our nanoparticles. These protein coronae will have a wide range of effects on our nanoparticles, like reduced infiltration ability. Secondly, we had to investigate isotopes and silver toxicity in particular. Furthermore, Cécile and Wessel advised us to look at the literature available on the RIVM website on nanoparticles, since the institution researches safety of nanoparticles as well.

Another point to investigate is the medical regulations in place. For this, our sources are European Medicines Agency (EMA) and the Zorginstituut Nederland (Dutch independent governing body for health care).

Lastly, it is essential to consider the practicalities of our product as a medical therapeutic. How, for example, will we discard the treatment-associated waste? This will be contaminated with nanoparticles so discarding it in regular waste will not be an option.

Interview 2

As mentioned previously, a second interview was held to give an update at the end of the lab time with Cécile. We discussed our progress on the safety topics.

In the meantime, RIVM had started the GREENER initiative²⁵. The GREENER initiative is regarding the sustainable production of medicine.

We inquired whether we would need to conform to these new initiatives, especially since the environmental biodegradability of our nanoparticles is still researched. Since, research is still being done, we cannot conclusively say whether our nanoparticles adhere to the GREENER criteria, therefore it is advised to continue development until more knowledge is available.

We also asked about the legally set stability requirements and whether coating will be another aspect to investigate when considering the disposal of nanoparticles. However, the answers to these questions are situational, so unfortunately, Cécile and Wessel were unable to help us further with this.

Supplementary literature

As advised, we investigated available literature on nanoparticles' effect on nature through RIVM. RIVM produced a simple model to predict the behavior of nanomaterials called SimpleBox4Nano²⁶. Some of the conclusions made by RIVM are that nanoparticles behave differently than traditional chemicals due to their tendency for agglomeration and dissolution²⁷. Further, short-term effects on the environment are limited, but long-term research is still required²⁸. For example, the study by Malejko et al.²⁹ did not observe short-term effects when investigating algal growth after 24-hour exposure to gold (III) nanoparticles coated with citrate. On the other hand, Brinkmann et al.³⁰ found that silver nanoparticles can cause acute immuno-toxic effects in vivo and in vitro. Part of the in vivo observations with silver nanoparticles can be attributed to the interactions between microbiota and nanoparticles and subsequent microbiota-host interactions³⁰.

Prof. Dr. Sylvestre Bonnet is a professor in chemistry at Leiden University. His group investigates the application of metal-based molecules in the fight against cancer and in the production of solar fuel. Due to the link between Prof Bonnet’s research and our project, we hoped that Prof. Dr. Bonnet could guide us more in our experimental design.

Integration

Prof. Dr. Bonnet is familiar with the possibilities we have at our lab at Leiden University, with his input we were able to create a final design for the Proof of Concept Experiments. Prof. Dr. Bonnet informed us the main parameter that matters for our nanoparticles is absorbance, meaning we had to use UV-vis to characterize our nanoparticles during our experiments and adept our gold and silver ratios as well as other components depending on the data we obtain thereof.

Interview

During our conversation with Prof. Dr. Sylvestre Bonnet a lot of the findings from previous literature research and the conversation with Dr. Dick Sterenborg were re-emphasized.

For instance, the absorbance of our nanoparticles will be dependent on their shape and composition. Therefore, it is important to first measure the absorbance of our nanoparticles using an absorbance measurement like UV-vis. Furthermore, Prof. Dr. Bonnet emphasized the importance of determining the composition of our nanoparticles, if they truly consist of silver and gold, and proposed that we could use his group’s Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Based on the absorbance measurement we have taken in our own lab, we could also identify a type of LED we would require to execute our photothermal experiments.

When discussing our ideas for a proof-of-concept experiment, Prof. Dr. Bonnet informed us that it is very ambitious to test our nanoparticles in tumor cells and not necessary. Therefore, he advises us to first execute some heating experiments where we excite our nanoparticles with the most suitable LED and then measure the increase in temperature using either a thermometer or an infrared heat camera. Furthermore, Prof. Dr. Bonnet offered to help us with these experiments since some people in his lab are well trained to work with lasers. To perform the experiments ourselves we would require some laser training.

Dr. Kristina Djanashvili is an associate professor in the group of Biocatalysis at Delft University of Technology. Her expertise lies in applied chemistry for imaging and therapeutic agents. One of our team members did an internship in her lab and from this we knew Dr. Djanashvili is working with nanoparticles, including their coating with various surfactants and functional molecules. The first conversation with her was to explore the possibilities of using our nanoparticles as theranostic agents, i.e. functional in both therapy and diagnosis.

Integration

Dr. Djanashvili highlighted the importance of using a coating. This will limit the precipitation of nanoparticles in the blood, limit corona formation and subsequent clearance through the immune system and will also limit the clumping of nanoparticles. If nanoparticles were to precipitate within the body, this will have detrimental effects on one’s survival. The lack of coating will also mean reduced effectiveness, so to be able to safely and effectively use our nanoparticles in a biomedical application, there is a need for coating.

In regard to targeting our nanoparticles, the Enhanced Permeability and Retention effect (EPR) will likely aid in the steering of our nanoparticles to tumors³¹. However, Dr. Djanashvili explained that opinions are still divided on this topic so we still investigated further targeting options for our Implementation. One point to consider in this is the size of the targeting agent and whether it will affect the surface chemistry of our nanoparticles and in the process the effectiveness of our nanoparticles as PTT. If we are unable to target our nanoparticles, there are little gains and we should focus on a different implementation. However, Dr. Djanashvili directed us to investigate how conjugating antibodies to gold nanoparticles actually increases Surface Plasmon Resonance (SPR) ³², the phenomenon that makes our nanoparticles usable in PTT. This indicates that by forming nanoparticle-antibody conjugates we could potentially increase effectiveness. This will hopefully also translate in requiring a lower dosage, limiting potential toxic side effects.

On the other side, however, is the requirement for antibody conjugation, that the pH of the solution is adjusted for antibody stability. Our modeling experiments showed nanoparticles are produced in a pH of 5. Literature showed that although silver nanoparticles are more stable at a pH of 8, they form smaller structures at lower pH. So, for our production process it is advisable to produce our nanoparticles at a lower pH and then stabilize them at a higher pH, where we will be able to bind antibodies for our final product.

Interview

During the interview Dr. Djanashvili has enlightened us further on the possibilities for a design of theranostics and the feasibility of the nanosystems we had in mind. The first aspect to consider is whether the diagnostic feature has an added value to our bimetallic particles that have been optimized to convert energy from a laser into heat. Depending on the imaging technique, some solid tumors can be visualized without the aid of imaging agents due to their high density. We would be able to use gold in our nanoparticles to create contrast in a CT scan. However, the right density of gold within the nanoparticles needs to be ensured in order to produce probes functional in CT. Furthermore, a coating of the surface of the nanoparticles needs to be considered.

We also brought up the topic of coatings during the conversation. A very interesting point made by Dr. Djanashvili was that we have to consider that nanoparticles have a relatively high density, and therefore, their precipitation cannot be excluded. If we were to inject nanoparticles into the bloodstream and they would precipitate at the site of injection, our nanoparticles would never work for PTT. Additionally, nanoparticles can accumulate in organs like the lungs and heart or block the small capillaries. Luckily, a well-chosen coating can improve the colloidal stability of the nanoparticles. Furthermore, a coating typically helps to prevent protein corona formation around the nanoparticles and therefore minimizes the chances for recognition of the nanoparticles by the immune system and their premature removal from the blood-circulation. An example of such surface molecules that Dr. Djanashvili works with and that could be an option for us is polyethylene glycol.

Another topic that came up was the targeted ability of our nanoparticles. Some of us were already acquainted with the Enhanced Permeability and Retention effect (EPR), which means that a targeting vector is not always required at the surface of a nanoparticle. However, the existence and magnitude of EPR are still under debate³¹. If we are to use a targeting vector, Dr. Djanashvili advised us to pay attention to the size of this molecule. Too large targeting vector could be difficult to install at the surface of the nanoparticle and its effect might decrease. She suggested not to limit ourselves to antibodies, but also consider other targeting vectors such as folic acid and small peptides, e.g. octreotate.

When asking about challenges during the development of theranostics, Dr. Djanashvili informed us that the first in vivo experiment often provides the first indication that the idea is practically feasible. However, it might take a long time until the first in vivo data can be collected. This has to do with the long administrative process around the ethical regulations in the partner laboratories. Of course they are there for a reason, but it does take some time to navigate through them.

Lastly, Dr. Djanashvili was able to give more insight into how to collaborate with companies. Usually, companies show interest in research that corresponds to their own vision and often do not have the resources and manpower to invest in projects beyond that. So, we would have to approach a company that has interests in line with our project. Furthermore, companies require regular updates from the research team and prefer to stay focused on the application rather than support a curiosity driven research. This may be perceived as a hindrance in the academic environment, but with the right industrial partner great things can be done.

Supplementary literature

Literature shows that gold nanoparticles can be used as a contrast agent in X-rays to visualize tumors^33,34. The golden standard currently in imaging head and neck cancers is CT³⁵. Since CT is composed of a collection of X-ray scans we can derive that the gold in our nanoparticles can have an additional imaging characteristic. Furthermore, there is the possibility of adding a fluorescent protein to the nanoparticles complex to foster NIR imaging³⁶.

To determine whether we will be able to use antibody-nanoparticles conjugates, we need to learn more about the effect of antibodies on the effectiveness of our nanoparticles in PTT. SPR and local SPR are highly variant depending on the surface structure and chemistry³². Lin et al.³² delineate that binding antibodies to metal nanoparticles could increase SPR locally and as a whole as well as increasing sensitivity. However, when using a nanoparticle-antibody conjugate, the solution should approximate that required for the stability of the antibody. Fernando and Zhou ³⁷ show that silver nanoparticles are more stable in a higher pH (pH of 8), but have a reduced size in a lower pH.

Camillo Iacometti is PhD candidate at the Institute of Biology Leiden. Mr. Iacometti’s expertise is metabolic engineering. We talked to Mr. Iacometti to optimize the synthetic biology aspect of our production design. In other words, we discussed the microorganism we use in our production design, E. coli, and our plasmid design to find out what would work for us.

Integration

Although we as a team are familiar with synthetic biology, we found it important to seek help to find the optimal microorganism and plasmids for our experiments. Mr. Iacometti has good experience in this regard and recommended using E. coli for our initial production design since it’s relatively easy to use. Furthermore, E. coli strains are typically fast growers (1 doubling / ~20 min) and this easily allows scaling up production compared to organisms like Deinococcus Radiodurans. Besides being widely used for producing pharmaceuticals, these features make E.coli a promising platform for the modern biotech industry. Mr. Iacometti hypothesizes that using low copy plasmids could be a safe choice: upon exposition to toxic metals, E. coli will likely increase the synthesis of proteins needed to cope with this stress; adding higher levels of protein expression could represent an excessive burden for the cell to grow smoothly. As we were unsure initially whether using a cell-free system (also have a look at our results) would work, we wanted to take into account the need to keep the E. coli alive during the nanoparticle synthesis. Therefore, to be sure we were able to produce nanoparticles, we took Mr. Iacometti’s advice and used a low copy plasmid.

Interview

During our conversation with Mr. Iacometti, he brought up what he calls his “bible”, a paper titled “An Engineering Approach for Rewiring Microbial Metabolism” by Wenk et al.⁶⁶ This article accurately describes principles and methods for establishing synthetic pathways into microbes and, according to Mr. Iacometti, it represents the right jumping off point to do “some cool synthetic biology in E.coli”. Mr. Iacometti mentioned that using this bacterium is a good decision since it is one of the most well-known and studied. Furthermore, a wide range of genetic tools are readily available for E.coli, meaning it can easily be engineered.

While discussing our plans for the project, Mr. Iacometti gave us valuable advice on constructing our plasmids for protein production using three levels of control. The first level, transcriptional regulation, is mediated by the promoter: a promoter can be classified as weak, medium, or strong based on its activity (strength), which determines how much mRNA is produced. Secondly, the ribosome binding site (RBS) is responsible for translational regulation and it defines how much protein is translated from the mRNA.

Last but not least, the origin of replication (ori) of the plasmid we use plays an important role in regulating the overall protein produced: e.g. a vector containing a high-copy number ori will be replicated many times (500-700 copies per cell), therefore huge amounts of the same protein will be expressed simultaneously.

Mr. Iacometti advised us to use a low-copy number plasmid to not stress out E. coli too much since we will already be stressing them out with metals. In our follow up conversation when asked how he would improve our experiments he said he would create a library of different combinations of plasmid copy number, different promoter strengths, and different RBS strengths for all our chosen genes. This would enable us to pinpoint the exact right combination to maximize our protein expression in order to produce the most nanoparticles.

Stefan Ellenbroek is the founding director of the life science and health incubator unlock_. unlock_ is based in Leiden and supports start-ups in the life science industry. Mr. Ellenbroek is an expert on the cross-section between financing and research in the life sciences. This conversation was an essential step to learning more about entrepreneurship and he guided us to reach out to more experts in various fields.

Integration

Mr. Ellenbroek recommended we reach out to DSM to learn more about upscaling and production, which we did. Also, Mr. Ellenbroek raised the concern of genotoxicity, which can be a determining factor for our proposed implementation. To investigate the possibility of genotoxicity and with that the safety of our nanoparticles we reached out to Paula van Rossum from Toxys. These conversations can be read later on the page.

Furthermore, Stefan Ellenbroek aided us in determining what kind of business models were feasible for us. He gave us the idea to work out a production process for our nanoparticles and leave the clinical trials to a different company or to proceed and work out the therapy aspect as well. The latter seemed very interesting to us at first, so we decided to investigate more about clinical trials to determine whether it would be feasible. However, as you can read on the Entrepreneurship page, we decided to go a different direction in the end after further investigation.

Interview

Mr. Ellenbroek informed us of the options that we have for Business models Entrepreneurship. Furthermore, it is important to be aware of the current market. For more insights, we should also look at current and past clinical trials. Information on these clinical trials needs to be publicly available according to the law.

Eventually, it will be important for us to convince stakeholders like healthcare authorities and the scientific community that our nanoparticles are better. For this, we need to know what distinguishes us from the market. What we eventually decided on can be read on Entrepreneurship.

Mr. Ellenbroek posed some interesting questions for us to look into. The first one is about the potential of cell toxicity and genotoxicity occurring due to our nanoparticles. For this, he referred us to talk to Paula van Rossum from Toxys.

Furthermore, Mr. Ellenbroek advised us to reach out to DSM to learn more about upscaling of production when dealing with bacteria.

Supplementary literature

There are a total of 182 clinical trials on thermal ablation, some of which have been completed³⁸. The pilot study done on head and neck cancers with Aurolase has been completed³⁹. In the results from the previously mentioned pilot, it can be read that there are some adverse events experienced.

Dr. Aquiles Carattino is a Co-founder and R&D Manager at Dispertech, a company that produces devices for nanoparticle analysis and visualization. Dr. Carattino obtained his doctorate at Leiden University. His dissertation was on the topic of metallic nanoparticles and how to detect them. In the conversation we had with him, our aim was to learn more about analysis techniques and the nanoparticles market.

Integration

Dr. Carattino re-emphasized that we need to investigate conformation change after heating in future experiments. We had already observed a shift in absorbance when we left our nanoparticles out. This conversation indicated that this is likely due to thermal instability recording dynamic absorbances. Literature indicates that coating can aid in thermal stability, however, we will still need to do experiments in the future to determine at which heating temperature our nanoparticles will lose their conformation. This finding did inspire us to try out different coatings to find an optimal one for our nanoparticles.

Unfortunately, we were unable to obtain access to the Nanoparticles Tracking Analysis (NTA) and Dynamic Light Scattering (DLS) during our lab time, which Dr. Carattino advised us to use. However, in future experiments, it would be very valuable to do analyzes with both techniques. Furthermore, it would be very interesting to try out nanoCET, the nanoparticle analyser produced by Dr. Carattino. This would also be an option to visualize our nanoparticles since they are present in liquid samples.

Interview

With this expertise Dr. Carattino could advise us on the analysis of our nanoparticles. He mentioned several machines and techniques that could be useful, such as NTA and DLS. We would be able to use these techniques to analyze the size of our nanoparticles.

Furthermore, he advised us that with a stable production protocol, metallic nanoparticles are unlikely to change. Therefore, nanoparticle measurements are only necessary occasionally to ensure similarity but are not required for every sample once a protocol is established. This is of great importance in upscaling, where controlling the product is of big importance.

Dr. Carattino’s company Dispertech, produces machines aiming to analyze and visualize nanoparticles. They have a machine, called nanoCET, which is able to measure nanoparticles in liquid samples and they are developing one for hydrogel samples. They currently provide their services mainly to researchers within institutions. The interest is still growing in this market, as the nanoparticles industry is relatively new.

Dr. Carattino was also able to share more of his knowledge on photothermal therapy. He informed us that there are two main methods used in photothermal therapy. The first one uses gradual heating, where you heat the nanoparticles to about 42°C for an extended period. The second method uses a shock method, where the aim is to heat up the nanoparticles for a very short time, up to 100°C.

When considering which type of photothermal therapy, Dr. Carattino also advised us to look at the conformation of our nanoparticles. Since we have protrusions/spikes, there is a chance that our nanoparticles will not be fully stable. This means that if we have a laser pointed at it for an extended period of time, the nanoparticles will deform to a more stable conformation like a sphere. This notion is in correspondence with what Prof. Dr. Dick Sterenborg has told us.

Supplementary literature

Literature shows that gold nanoparticles are not thermally stable. Gold urchin-like nanoparticles fall back to the more favorable conformation, sphere, during heating^40,41. Some even lose stability at room temperature, luckily capping can aid in improving stability⁴². However, Petrova et al.⁵⁵ found that when heating up gold nanorods using a laser, conformation was retained during heating up to 700°C.

Aaike van Vught is the CEO of the start-up Vsparticle, a company focused on producing nanoparticles for industry. Vsparticle has produced a NanoPrinter, a machine able to print nanomaterials for application in industries like energy storage. With this conversation, we hoped to learn more about entrepreneurship and the nanoparticles market.

Integration

Mr. van Vught told us there are plenty of other applications for nanoparticles. Once we have an established method of production, we would be able to investigate other applications and widen our market share by investigating potential applications in air sensors and energy storage as Mr. van Vught delineated.

Furthermore, from our conversation we realized that it is important to sterilize our nanoparticles for biomedical applications. This is possible using filter sterilizing, which we have implemented into our experiments. However, we could investigate other options in the future, since we observed a change in absorbance when using filter sterilization, see Results page.

This conversation with Mr. van Vught enabled us to understand more of the nanoparticles and head and neck cancer treatment market. The Tam Sam Som analysis was an essential part of our entrepreneurship.

Interview

Since Mr. van Vught has set-up his own company, he was of big help when discussing entrepreneurship. He directed us to some methods on how to sell our idea. One method he explained was the Tam Sam Som go-to-market strategy. To pursue a potential start-up, we should get a focus group for our project to obtain quick feedback on our product. To obtain a focus group and to gather interest into our project we should visualize our intentions well. Mr. van Vught also aided in our outreach by giving suggestions on marketing.

Furthermore, we have been discussing the potential of our nanoparticles to be functional in other fields. Mr. van Vught delineated some fields where there is increased attention for nanoparticles. The first one being the energy market, where nanoparticles could function as a stabilizing factor in the packaging of hydrogen fuel. With the current state of the gas market and the increased environmental consciousness, this is a very interesting market to investigate in the future. A second option is the increased interest for nanoparticles in air sensors to aid air purification by gas adsorption, bacteria disinfection and particle filtration, something that has been popular since the corona pandemic.

Another topic of high interest for us is environmental safety. At Vsparticles they have been able to account for this by building a closed system. We have to investigate whether this is possible when looking at up-scaling. Furthermore, Mr. van Vught advised us to review the research done by the Rijksinstituut voor Volksgezondheid en Milieu (RIVM), National Institute for Public Health and the Environment) with regard to environmental safety and nanoparticles.

Another point that Mr. van Vught made - and where they are more advanced in - is to investigate methods to “clean” our nanoparticles. We should get rid of cell debris, proteins and contaminants, which could potentially be a hard task.

Lastly, Mr. Vught advised us to reach out to some interesting stakeholders, like the company Ginkgo Bioworks and venture capitalists in the future.

Supplementary literature

Sterilization of nanoparticles is essential when producing them for biomedical purposes. Some sterilization techniques can alter characteristics like size, polydispersity index, and stability. There are some sterilization techniques available like sterile filtration, UV radiation, and autoclaving⁴⁴. We will have to validate which sterilization technique is most suitable for our nanoparticles in the future, however, we have been using sterile filtration already during our experiments, see Results page.

Dr. Anke Hövels is the strategic lead of Access to Medicine at the Dutch Cancer Society (KWF). She is an expert in market access of pharmaceutical treatment, with years of industry and academic experience. From our conversation, we hoped to learn more about the economical side of developing cancer treatments, head and neck cancers in a larger framework, and important considerations for future work with our nanoparticles.

Integration

During our conversations with Dr. Hövels we learned a bit more about our proposed implementation of using our nanoparticles in treating nanoparticles. Recent studies have shown the need for more attention to rare cancers like head and neck cancer, which is part of our project. It shows the social impact that our project has by helping a vulnerable group in society.

Furthermore, we gained a better insight into the Dutch market system for new cancer therapies. Dr. Hövels advised us to push the therapy to head and neck surgeons instead of hospital administration. This is integrated into our business model, see Entrepreneurship page.

Also, it was confirmed that specificity is important when developing new treatments.

It was also mentioned that we should have contact with the European Medicines Agency (EMA) in the early stages of treatment development. Luckily, we already have had some contact with both the EMA and Rijksinstituut voor Volksgezondheid en Milieu (RIVM, National Institute for Public Health and the Environment) about regulations regarding medicine development and the production process. In the future, we would have to have a lot more contact with the EMA and RIVM, so it might be wise to get help from an expert in this area.

Interview

Rare cancers are often overlooked

Rare cancers were an important topic during our conversation. Before having this interview, we had already decided to focus on head and neck cancers. Even though head and neck cancer is common (over 3000 patients a year in the Netherlands), the cancers that fall within this category, such as mouth cancer or laryngeal cancer, are all rare. This means that less than 6 for every 100,000 people are diagnosed in a year. the Netherlands⁴⁵.

Dr. Hövels informed us of a recent study by de Heus et al.⁴⁵ that found patients with a rare cancer are behind in survival. It found that there have been no reduced improvements in survival compared to common cancers. This is especially worrying, as 18% of all adults are diagnosed with a type of rare cancer (excluding hematological and childhood cancers)². The KWF, therefore, strives to support research in this area. Dr. Hövels mentioned that an essential - but often overlooked - part of research is knowing what type of cancer you want to target. Since we already know our intended implementation, we are on the right track.

A need for more specific treatment

When we inquired about desirable aspects of newly developed therapies, Dr. Hövels agreed with the doctors we spoke to previously: specificity is key. For a long time, major side-effects were accepted in treatments, since cancer is a disease with a devastating impact. However, in recent years the focus on a good quality of life during and after treatment has increased. This shift also demands a more precise cancer treatment. The Commission Bom advises the Zorginstituut on improvements in this area⁴⁶.

Bringing our therapy to the market

Dr. Hövels emphasized that it is important to get into contact with regulatory bodies like the EMA early in the development of a therapy. Therapies are only accepted if there is Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP). You will have to create a lot of paperwork but will aid in later stages of development by preventing you from having gaps. Advice Dr. Hövels gave, is to hire a regulatory consultant. Scientific advice from the EMA about the foreseen production processes is also important.

The final step after the therapy is approved by EMA, is to bring it into practice. To make the therapy usable and interesting for hospitals, it is advisable to get expertise from specialists in the medical area. These specialists can be involved in the clinical trials and research before clinical trials, but can also be contacted at congresses. Once specialists have faith in the therapy, it will be passed on to hospital purchasing agents who will be the ones buying the therapies for hospitals. Eventually, it can then be used to treat patients.

Supplementary literature

The publication mentioned in the conversation shows that there is a need for better diagnosis, novel therapies, establishing expertise centers and more research to better survival of rare cancers⁴⁵. In the process of reading that publication, we came across another about occupational rehabilitation for rare cancer patients. It showed that there is a need for more experience and knowledge here as well. Raising awareness and providing personalized care will better the situation significantly⁴⁸.

Paula van Rossum is chief business officer at Toxys. Toxys is a spin-off of the medical center in Leiden (LUMC). They provide in vitro testing for (geno)toxicity, always focused on helping to understand the mode-of-action of toxic compounds. These tests are not required by law, but will give more insight into the toxic mode-of-action. For ToxTracker, a genotoxicity assay, they use undifferentiated mouse stem cells and are able to identify over 95% of all genotoxic chemicals. Their specialized set-up called Toxtracker is able to identify mode of action of toxic chemicals. We aimed to learn more about the toxicities of nanoparticles.

Integration

Toxicity is an important aspect of any medical product. Of course, we aim to produce a product that is the least toxic as possible. Learning more about the toxicities associated with nanoparticles showed how diverse toxicities are depending on their characteristics (like size and surface chemistry) and what experiments are used to test this. Therefore, we learned that we cannot rely on publications of other nanoparticles and will have to test our nanoparticles according to the regulations set-out by European Medicines Agency (EMA) as well as additional toxicology tests to get a comprehensive overview of toxicology.

An essential part of potential toxicology is whether coating will be persistent. Due to heating, the coating can alter form or become leaky. If this is the case, this can both improve and decrease the safety of our nanoparticles. It can improve it, by enabling clearance through the immune system as we discovered from literature research after speaking to Dr. Kristina Djanashvili. However, it also can mean a greater toxicity. This will be an essential part for future experiments, see Implementation page, as well as prompting us to not be too focused on the toxicity of other nanoparticles.

Interview

We decided to speak to Ms. van Rossum based on the interview with Stefan Ellenbroek.

Ms. van Rossum explained to us that the chance of genotoxicity is dependent on the type of material, meaning we have to investigate gold and silver. More important for our project, however, is to investigate viability and potential cytotoxicity like oxidative stress. This is usually prominent when using metals. The typical damage observed in ToxTracker for nanoparticles is related to oxidative stress. To learn more about this Ms. van Rossum advised us to read some of the publications by the Karolinska Institutet.

Ms. van Rossum expects that our coating will eventually dissolve since otherwise it could hamper the functionality of our nanoparticles. Therefore, she advises us to test our nanoparticles both with coating and without coating for toxicity. Furthermore, based on her experience she expects that the shape of our nanoparticles will not necessarily cause genotoxicity. However, size could be a determining factor for genotoxicity.

An aid in determining toxicities is to create a hazard assessment. This will indicate the intrinsic potential to be harmful. Later on we will have to produce a risk assessment with indications of what is a safe dosage.

Supplementary literature

Bulk gold is known to be a safe material. However, the size of nanoparticles brings about unique properties that can cause toxicities, of which nonspecific oxidation caused by reactive oxygen species is of most concern⁴⁹. Most studies on genotoxicity show positive results for gold nanoparticles, however some have shown negative results². These differences are dependent on nanoparticle size, the presence of coating, different concentrations and many more². For example an in vivo study using the SMART test with gold nanoparticles showed a higher genotoxicity in 90 nm nanoparticles compared to smaller ones⁴⁹. Further, Sowmiya et al.⁵⁰ showed concentration dependent genotoxicity of nanoparticles in zebrafish.

Gliga et al.⁵¹ showed that silver nanoparticles have size dependent cytotoxicity.

In regard to bimetallic nanoparticles, Padmos et al.⁵² have shown that adding gold to silver will lower the toxicity of silver in mammalian cells.

To conclude, there is a concern that our nanoparticles can be toxic. However, the European Commision has stated that toxicity of nanoparticles will be tested case specific⁵¹. Therefore, we will be doing toxicology testing in the future. Of course toxicology tests are required in all medical applications so it was already accounted for in our future plans.

Prof. Henk Noorman is a part-time professor in bioprocess design and integration at Delft University of Technology. He also works at DSM as Senior Science Fellow. Prof. Noorman is an expert in bioprocesses and upscaling. With this interview, we aimed to learn more about the possibilities of upscaling and worker safety and to get feedback on our intended production process.

Integration

This conversation ties into the conversations we have held with Rijksinstituut voor Volksgezondheid en Milieu (RIVM, National Institute for Public Health and the Environment) and Stefan Ellenbroek. Dr. Noorman gave us a better idea of how we will be able to upscale our production and what experiments we still have to execute to find the right concentrations for components like levels of oxygen and silver and gold. Further, a better idea of a method for isolating our nanoparticles on a large scale was given, namely ultra-centrifuge or nano-centrifuge depending on density. Strategies to improve even more on one of our keyvalues, environmental friendliness, were also discussed, like obtaining glucose that is ethically and environmentally friendly sourced.

Lastly, it is feasible to set up a large-scale production process for our nanoparticles in terms of regulations we will need to uphold. However, Dr. Noorman warned us that we should be cautious that redox reactions need a cell wall to produce sufficient energy input. Meaning that our current production design might need adjusting to foster continuous redox reactions by not lysing the cells, or to find a different solution to ensure plenty of available energy. However, this would also mean we are unable to use high concentrations of gold and silver since that will stress the bacterial cells too much. So, we will either have to prioritize either less efficient production with a higher number of nanoparticles produced or a more efficient production with a lower amount of nanoparticles produced. Part of this consideration is also the safety (using living Genetically modified organisms (GMOs) or cell-free system) and environment friendliness ( bioreactor produces one batch or multiple batches of nanoparticles). This all ties in with our entrepreneurship and specifically our SWOT analysis, see Entrepreneurship page.

Interview

The primary question for this interview was about how we would be able to upscale. We have spoken about this in earlier interviews. There are a couple of options for upscaling. However, since we have quite a specific purpose for production, we would be able to produce on a relatively small scale. Prof. Noorman advised us to think about a bioreactor with a size of about 1000 L. We could produce a very roughly estimated 1 kg of nanoparticles from this. For this, we would be able to use single-use bioreactors from an equipment supplier such as Sartorius or Thermo Fisher. This has the benefit that we will not have costs to clean out and sterilize the reactor. However, additional costs of waste disposal need to be factored-in.

To be able to know more about the scalability of our nanoparticle production, we should do first experiments at about 10 L, i.e. beyond the micro-titer level. According to Prof. Noorman, production at this scale is reasonably similar to that at 1000 L, and we would need to make only modest adjustments upon scale-up. Still, we will likely have a relatively decreased production in a bioreactor compared to a plate.

We should ensure that a chemical redox reaction can take place, in order to regenerate the biological co-factors used in the enzyme reactions.

To be able to isolate our nanoparticles from the bioreactor, Prof. Noorman advised us to look into the density of our nanoparticles. Based on this knowledge we will likely be able to use a filter or centrifuge..

To learn more about sustainability, Prof. Noorman referred to the use of renewable energy for the entire process, and to minimize waste production. Any remaining waste, or co-products, should have a certified outlet, e.g. via plastic recycle or waste (water) treatment. Also, raw materials should be sourced with the lowest carbon footprint.

To learn a bit more about worker protection - like we were advised by the RIVM - we inquired about this. One important aspect is to have clear protocols for employees and to use multiple levels of containment, closed systems,whenever possible. For example, working with a GMO requires strict protocols and a closed sewage system just like in hospitals to prevent contamination and leakage to the environment. Lastly, there is also external supervision that is licensed out by RIVM.

Luckily, our production process mainly is based on combining existing technologies in a new way, which means that we will have relatively fewer trouble with regulations even though we still need regulatory approval.

Dr. Sylvia van Egmond is head and neck surgeon at Leiden University Medical center (LUMC). Dr. Martin de Jong is a radiotherapist at LUMC. They cooperate when treating patients with head and neck cancer. This conversation was held to gain knowledge about current practices and the potential of PTT in the future.

Integration

As an answer to our moral questions regarding the use of metals and Genetically modified organisms (GMOs) for medicinal purposes, Dr. van Egmond and Dr. de Jong also expect that no big ethical questions by patients will be raised due to our nanoparticles. Of course, this is still subjected to personal opinion but in a general sense, there are no big ethical concerns. This is largely due to a readily accepted use of GMOs for biomedical production purposes and the general perception that gold is especially safe to use in biomedical applications, which led us to continue with these components in our product design.

Dr. van Egmond and Dr. de Jong emphasized that precision therapy is desirable, consequently important to have a social impact. This means that for our implementation, a targeting technique is important. Luckily, we have been investigating the possibility of using antibodies (see the conversation with Mr. Dimitri Pappaioannou) as a targeting mechanism previously.

Further, we learned a lot about the current status of treatment so we are more aware of what to compare PTT to which is beneficial for our Entrepreneurship.

Interview

When asked whether Dr. van Egmond and Dr. de Jong are familiar with PTT they responded in the negative. The most common treatments used are a combination of surgery and radiotherapy. Currently, if they find that the edges are not “clean”, they will radiate the whole surgery region. However, since a tumor never has a homogenous shape, this means you are also radiating healthy tissue. Radiation, however, has the benefit that you are not removing any additional tissue. Every time, they aim to treat as least as possible with maximal results.

When inquiring about the potential advances that should be made when treating head and neck cancers, Dr. van Egmond and Dr. de Jong responded that there are three main improvements to be made.

The first one is a desire for more specificity. The head and neck region is highly functional and any tissue that you are able to spare, you want to spare. Especially after previous treatment, it can be very difficult to identify the boundaries of a tumor.

Secondly, is to have a better way of visualizing where the tumor and its branches are situated. There are currently some studies investigating the use of dyes for imaging to improve this visualization. As mentioned before, the head and neck region is highly functional, so any type of treatment will impact quality of life.

Thirdly, the reliability of treatment is also very important. Some questions posed by Dr. van Egmond and Dr. de Jong are whether our nanoparticles will be able to infiltrate a bulk tumor and whether the NIR laser will be able to excite nanoparticles throughout the bulk tumor. If this is not the case it could indicate decreased reliability and the need for repetitive treatment.

When inquiring whether we could expect any reservations regarding the use of GMOs in the production of our nanoparticles and the fact that we will need to inject metals into someone’s body. Dr. de Jong and Dr. van Egmond responded that this will be dependent on one’s own opinions. You will have people willing to accept this, but there are also people who would not.

Lastly, Dr. van Egmond and Dr. de Jong advised us to read up on hyperthermia, a cancer treatment also using heat.

Supplementary literature

Hyperthermia is a form of cancer therapy where tissue is heated. There is the option of regional or whole body hyperthermia. Hyperthermia is an adjuvant therapy and is usually given with either radiotherapy or chemotherapy⁵³. Some of the benefits are increased blood circulation, hypoxia, and influencing immunogenicity within tumors^54,55.In pancreatic cancer, hyperthermia can positively influence treatment outcomes although further research needs to be done on the topic⁵⁶.In head and neck cancers, there have been better results when radiotherapy and chemotherapy have been combined with hyperthermia.⁵⁵ Magnetic nanoparticles have even been used as hyperthermia agents in research and show promising results^55,57.In fact, PTT can be used as a form of hyperthermia that is more localized⁵⁸.

In addition to the previously held conversations with Dr. van Egmond and Dr. de Jong about the current practices of treating head and neck cancers, we also had a conversation with Prof. Dr. Remco de Bree. Prof. Dr. de Bree is a professor and head of the department of Head and Neck Surgical Oncology at University Medical Center Utrecht (UMCU). His expertise lies in diagnostic imaging techniques. The goal of this interview was to learn more about current practices, the potential of diagnostics, and the potential of PTT in the future.

Integration

Dr. de Bree pointed out the importance of nanoparticle dispersion as an essential aspect of PTT. Literature shows that dispersion in the periphery is sufficient to treat thin tumors (< 6 cm). Head and neck tumors are usually only a few centimeters thick. This means we do not need to have concerns regarding the size of our nanoparticles and whether they would be able to infiltrate well throughout the tumor matrix.

Lastly, there are indications from literature that healthy tissue will not be as sensitive to the temperature increase mediated by our nanoparticles due to the different properties of the tissue. This means that even if our nanoparticles are present in healthy tissue, they will likely be less harmful than in tumor tissue. Meaning that using our nanoparticles will likely be a safer treatment compared to radiotherapy.

Interview

Currently, the main therapies in use for treating head and neck cancers at the UMCU are surgery, radiotherapy and/or cisplatin as chemotherapy. Even though in the last few decades, radiation and surgery have improved (with the latter a better ability to reconstruct), the side effects are usually a determining factor of which primary therapy to use. Radiation in the mouth causes a lot of side effects, so usually, surgery is applied in that area. On the other hand, in the oropharynx area, it is difficult to perform surgery and you would need to create a different access point to be able to operate on the whole tumor, so then preference is given to radiotherapy. There are ongoing trials with using systemic treatments alone like chemotherapies and immunotherapies, however not in the curative setting yet.

Most tumors in the head and neck region are a result of either smoking and drinking or a hereditary disposition. This means that there often will be a second or third primary tumor. These additional primary tumors are usually increasingly difficult to treat. This is due to damage done to the area by previous treatments, treating the same way will kill too much healthy tissue.

However, what is essential according to Prof. Dr. de Bree is better diagnostics. In diagnostics, you are able to be more personalized. With the personalization of therapy, you can talk about specific markers of a tumor, but personalization starts before this.

Whether you will overtreat or undertreat patients is highly dependent on diagnostics. Now mainly overtreat because we are afraid to undertreat enough, however, this means that you will have to give up some quality of life. Therefore it is important to better diagnose metastasis in the future, including distant metastasis. If you are able to better predict whether there are metastases, then you can provide more tailored treatment.

When asked for his opinion on the application of our nanoparticles as a diagnostic tool in addition to a therapeutic tool, Prof. Dr. de Bree answered that it is interesting. A method like we have the potential of producing we would be able to have insights in what is happening when giving the treatment. They are currently experimenting with better ways to visualize sentinel nodes a tumor drains to, to improve visualizing potential metastasis. Early visualization aid in the prevention of more extensive metastasis formation. There is currently research ongoing on the use of iron nanoparticles and MRI to aid in sentinel node visualization.

Furthermore, after having explained some of the considerations we are taking in regard to attaining specificity, Prof. Dr. de Bree noted that we would need to have good antibodies. In head and neck cancer there is relatively less clinical investigation into markers.

When inquiring about Prof. Dr. de Bree’s opinion of Photodynamic therapy (PDT), he answered that there are possibilities for using alternative types of therapy like PDT and PTT, but preference is given to the standard methods that a practitioner is well acquainted with. There are some challenges we will have to overcome, like when using a laser for treatment, the tumor needs to be accessible to the laser and should be less than a cm thick. PDT is also very impactful for a patient since there are quite some rules they have to follow. Dr. de Bree views PDT as it is now as a last option or specifically for tumors that are quite large but thin.

Lastly, Prof. Dr. de Bree posed some very important questions. The first one being: Do we need our nanoparticles to be in contact with every tumor cell to be functional? It is also important to distinguish between healthy and tumor tissue. Another important point to consider within this consideration is that in the head and neck region there are a lot of major arteries that cool down the tissue. This means that a therapy like hyperthermia and PTT may have reduced effects due to a reduced ability to increase tissue temperature.

Supplementary literature

Direct injection of nanoparticles within a tumor shows an asymmetrical spatio-temporal temperature field⁵⁹. Meaning we would need good dispersion of our nanoparticles within the tumor tissue. Soni et al.⁵⁹ found that accumulation of nanoparticles at the periphery - as is expected to occur when nanoparticles are injected intravenously - is more effective compared to uniform distribution within the tumor and confined to one area within the tumor - as when injecting right into the tumor.

Furthermore, vasculature is one of the determining factors for thermal ablation to work⁶⁰. Luckily, the preliminary study done by Belfiore et al.⁶¹ shows promising results for using thermal ablation in different types of head and neck cancer where tumors are < 6 cm.

Regarding the distinction between healthy and tumorous tissue, there is no real difference in thermal sensitivity between tumorous and normal tissue. Although the exact mechanisms are unknown it is observed in vivo that tumorous tissue is more likely to die. A likely explanation why tumor tissues are more sensitive to hyperthermia is due to their unstructured vasculature which in its turn causes a low pH and hypoxia^62,63. Beik et al.⁶⁴ note a study that tested monoclonal antibodies conjugated to gold nanoparticles which showed no significant damage to healthy cells.

As was mentioned before, healthcare coverage can be an important factor for our treatment to be implemented in practice. In the conversation with Dr. Anke Hövels, we were directed toward Dr. Geert Frederix. Dr. Geert Frederix is an Associate professor in Health Technology Assessment at the Department of Public Health, Healthcare Innovation & Evaluation, and Medical Humanities at University Medical Center Utrecht (UMCU). He researches bio-medical innovations, market access, and economic evaluations, with a specific focus on diagnostic tools. The conversation was to learn more about how we would be able to obtain coverage for our nanoparticles as PTT, what is included in an economic evaluation and when an economic evaluation should be done.

Integration

Whether our nanoparticles can be successfully implemented as a treatment, is highly dependent on whether it can be used in practice, both in regards of science and finance. If no coverage by health insurance can be obtained, it will be difficult to succeed with the implementation as PTT. A step we could then take is to try and lower production costs. This can be achieved by reducing costs of raw material (i.e., gold and silver) or reducing energy costs (i.e. producing only on demand). What is most likely, however, is that we will focus on a different implementation (i.e., using as a connecting structure in nanoparticles).

Our nanoparticles on their own do not qualify as a medical treatment, however, Dr. Frederix explained that whether our nanoparticles as PTT will be covered by healthcare is dependent on the economic evaluation we will have to produce ourselves during phase 3 clinical trials. The economic evaluation records aspects like survival rate, cost of hospitalization, and time of incapacitation. These economic evaluations are usually also required by the European Medicines Agency (EMA). In addition, as Dr. Frederix told us, an early economic evaluation can be used to inform potential investors and is, therefore, an essential part of our Entrepreneurship.

Interview

Dr. Frederix informed us that during phase 3 clinical trials, an economic evaluation is made which includes a reimbursement file and clinical efficacy. This evaluation compares the new therapy to existing therapies to see what improvements are made and in which areas. A good way of doing a comparison is to investigate survival studies. Furthermore factors like cost of hospitalization, additional medical costs, and the time a person is incapacitated influence the economic evaluation.

Since economic evaluations are predictions for the future, there is still a lot unknown. Therefore, as we are doing right now, stakeholders and experts are contacted. Additionally, assumptions are made, for which it is important to remain transparent.

These economic evaluations are usually very similar to what is required by EMA. In the Netherlands, this document is given to the Zorginstituut Nederland. There they evaluate the application and advise the minister about approval of the application, after which the minister has final say.

When a new therapy is adopted in the Zorginstituut Nederland, it is re-evaluated once every couple of years depending on how expensive it is.

Big pharma companies have specialized departments that focus on market access. These departments evaluate and produce economic evaluations.

The current practice is to execute these economic evaluations when quite a lot of data is already produced. Recently, however, Oncode - an independent institute involved in improving cancer research and translating research to implementation - has been doing these economic evaluations earlier in the research process. This ensures that projects likely to be non-profitable are filtered out. Furthermore, it can be used to inform investors and researchers whether there is enough potential for improvement.

When talking about the potential theranostic properties of our nanoparticles, Dr. Frederix informed us to see diagnostics as a separate component completely. We would have to register for both therapy and diagnostics separately, so combining the two will not lead to less work.

Supplementary literature

In the Netherlands, there is a database where all incidence and mortality of cancer is recorded. For head and neck cancer, 921 people who were given the diagnosis died in 2020⁶⁵. When this is corrected with the increase and aging of the population, 3.32 people out of 100,000 people passed away to head and neck cancer⁶⁵.

Head and neck cancer patients are the primary stakeholder for our project. After initial communication with (ex-)patients we learned about their difficult experiences with cancer and rehabilitation. Hereafter, we refrained from conducting interviews based on ethical considerations,as we are not trained professionally in this aspect. Another option was to send out questionnaires, but we felt that this could activate the (ex-)patients’ trauma as well. Alternatively, we could review literature and gather experience-based information from doctors which we prefered as this option did not burden patients further.

Integration

From what is described in the literature, we can accept there are a lot of adverse effects associated with head and neck cancer treatment. A better specificity of treatment by using targeting techniques will lessen the side effects experienced and will improve quality of life of head and neck cancer patients which is morally desirable. Selectivity of thermal treatment and with that less experienced side effects can be obtained using nanoparticles like ours,⁵⁹ showing the social impact the production of our nanoparticles has. Additionally, PTT has a high specificity and minimal invasiveness compared to current treatments used⁶⁷. Therefore, our project takes a step in the direction of bettering treatment.

Literature

One of the main adverse effects of current head and neck cancer treatment is oral dysfunction with associated speech and swallowing impairments⁶⁸. The NET-QUBIC study, a cohort study for head and neck cancer patients, found speech deficits in 46.1% and moderate-to-severe experienced neurocognitive deficit in 4-8% of the patients⁶⁹. Persistent bad sleep was experienced by 21.8% of head and neck cancer patients 6 months after treatment, with 10.9% experiencing a decrease in quality sleep during the 6 months after treatment⁷⁰. The same NET-QUBIC data shows that 53% of the patients experience swallowing problems prior to treatment, this increases to 70% during the first 3 months after treatment and then decreases again to 50% at 1 year post treatment⁷¹ and 52.8% of patients experience a high fear of cancer recurrence⁷². In line with these adverse effects, many patients experience malnutrition due to loss of appetite or problems with eating⁷³. A common comorbidity is depression⁷⁴.

Up to 75% of head and neck cancer patients treated with surgery experience body image issues⁷⁵. Hearing loss can be experienced in patients treated with radiotherapy and/or cisplatin⁷⁶. Patients who have undergone radiotherapy have additional late side effects like dental and oral problems, structural damage to important features in the head and neck region and more⁷⁷. Adverse effects experienced when treated with photodynamic therapy (PDT) are pain after treatment, burning or prickling sensation, and photosensitivity⁷⁸.