Overview of Binanox

Nanotherapeutics are rapidly emerging in the field of head and neck cancer therapy as researchers work to develop treatments that are highly targeted, less invasive, and tunable^1,2,3. Current standard treatments such as radiotherapy, chemotherapy, and surgical resection can be limiting due to toxic post-treatment side effects, increasing resistance, and treatment failure^4,5,6. These factors show an urgent need to develop effective therapies to ensure that a patient's quality of life is not compromised. Photothermal therapy (PTT) is a novel approach utilizing near-infrared (NIR) light absorbents capable of converting NIR light to heat. The produced heat ablates cancerous cells in a process referred to as photo-hyperthermia^1,6 . Metallic nanoparticles, for example made of gold (Au) or silver (Ag), are suitable agents for PTT and effective NIR absorbents. Their ability to absorb NIR light can be further enhanced by modifying the composition and shape of the nanoparticles^1,7,8,9. With that in mind, our project aimed to produce urchin-like, bimetallic nanoparticles composed of a silver core and golden spikes. We aimed to utilize a microbial factory designed by genetically modifying Escherichia coli to express metal-reducing proteins obtained from Candida albicans, and from a metal-resistant bacterium, Cupriavidus metallidurans^10,11,12. By attempting to biologically produce and modify the structure of these metallic nanoparticles, we hope to establish a platform dedicated to optimizing PTT, a promising therapeutic approach only achievable through nanotechnology.

Current trends in photothermal therapy and how can we improve it?

PTT is a therapeutic approach in which NIR light is converted to thermal energy to kill cancerous cells by using photothermal agents¹³. Metallic nanoparticles serve as an ideal photothermal agent due to a unique, intrinsic property referred to as surface plasmon resonance (SPR)¹⁴. Here, the particles absorb photons from the light wave, causing electrons within the metal to oscillate at a frequency identical to this light wave. This results in the production of energy that is emitted as localized heat, which can kill cancer cells when the particles are in close proximity to the solid tumor (Fig. 1)^15,16. The specificity of these nanoparticles to the tumor can be further enhanced by adding tumor specific antibodies¹⁷. Read our Proof of Concept page where we test whether nanoparticles produced in our laboratory possess SPR properties.

Current research trends in metal-based PTT are mostly based on investigating the potential of gold nanoparticles in cancer therapeutics¹⁸. It has been demonstrated that nanoparticles with an uneven surface, particularly urchin-like gold nanoparticles, have an increased surface plasmon resonance and absorb light between wavelengths of 750-900 nm (Fig. 2)^8,18,19 . This suggests that these particles may potentially be more effective agents for PTT, because this wavelength can penetrate human tissue.

Thus, inspired by this novel field aimed at improving PTT, our team took up the challenge to identify features that may further enhance the properties of these nanoparticles. Furthermore, we aimed to use a microbial system to achieve the biological production of these metallic nanoparticles optimal for PTT. We identified three main features, namely (i) bimetallic, (ii) urchin-like nanoparticles, and (iii) employing particles within the size range of 20 and 150 nm . The role of these features in improving PTT effectiveness is further detailed below. You can also check our Safety and Integrated Human Practices page to read about other aspects we explored, ranging from toxicity to increasing specificity of nanoparticles to tumor tissue.

Bimetallic nanoparticles: the best of both worlds

As noted earlier, in addition to SPR, urchin-like gold nanoparticles show absorbance peaks between 700-900 nm¹⁹. This is a suitable absorption range for PTT since this wavelength can penetrate human tissue, making PTT a non-invasive approach²¹. Furthermore, gold is highly photostable and has a low cytotoxicity²². Besides monometallic gold nanoparticles, evidence suggests that silver nanoparticles possess the highest SPR properties when exposed to NIR^15,20. Moreover, silver nanoparticles have shown antitumor properties by disrupting structure and function in cancer cells¹. These include the production of reactive oxygen species that can trigger apoptosis, disrupt protein function, block antioxidants that may eliminate ROS agents, and disrupt energy pathways by interfering with mitochondrial function¹. To combine these advantages suitable to PTT, our team decided to produce bimetallic nanoparticles composed of gold and silver.

Why urchin-like?

The morphology and composition of the nanoparticles greatly influence nanoparticle effectivity¹⁸. The golden spikes in urchin-like nanoparticles have a smaller radius at the ends, which enhances the surface plasmon resonance at the tips of the spikes by enhancing the electromagnetic field^{18, 23}. Thus, to combine the efficient light-to-heat energy conversion of the golden spikes and the antitumor properties of silver, we decided to biologically synthesize nanoparticles with a silver core and golden spikes.

Size of nanoparticles?

(Adapted from the MSP literature review by iGEM Leiden 2022- Collaborations)
Nanoparticle size affects how suitable the particles are for PTT. Nanoparticles larger than 200 nm get removed by the reticuloendothelial system, which causes quick clearance and, thereby an ineffective therapy²⁴. Research suggested that filtration by the reticuloendothelial system can be avoided by using nanoparticles smaller than 150 nm^24,25. However, nanoparticles of a size under 10 nm, are also unsuitable for PTT, since they quickly get filtered out of the body by the renal system due to their small size²⁶.

Additionally, accumulation of nanoparticles at sites outside of tumor tissue is undesirable since this can have cytotoxic effects. Investigations show that nanoparticles of 50 nm in size have a low organ accumulation^27,28. Another factor that influences the efficiency of PTT is how well the particles are able to reach the tumor cells. Due to the leaky vasculature and the poor lymphatic drainage around tumors, the interstitial fluid pressure in tumors is high. Studies have shown that nanoparticles of 12 nm and 20 nm are most effective at entering tumors but have a lower heating capacity^29,30,31,32. Hence, the range for nanoparticles suitable for PTT lies between 20 and 150 nm, and within this range certain sizes are more favorable for specific aspects. For example, while nanoparticles of 50 nm show low accumulation in the liver and spleen, nanoparticles around 20 nm are best at reaching tumor tissue. Therefore, we aimed to produce nanoparticles between 20 nm and 150 nm.

Binanox: a microbial factory for bimetallic nanoparticles

Bacterial cells are able to reduce metallic ions to their elemental forms as a survival strategy, since metal ions can be toxic to bacterial cells³². At Binanox, we aimed to enhance this naturally occurring system to produce urchin-like bimetallic nanoparticles optimal for PTT. We used synthetic biology to engineer Escherichia coli (E. coli) with metal-reducing genes obtained from other organisms more efficient at metal ion reduction. Interestingly, literature has shown that microbial systems can be suitable for the biological production of urchin-like bimetallic nanoparticles with a silver core and golden spikes³³. For example, Chen et al. proved the formation of jagged bimetallic nanoparticles using Deinococcus radiodurans³³. Quite remarkably, these nanoparticles were produced using a cell-free system, which suggests that the produced nanoparticles are free of GMOs.

Thus, inspired by this field of research, we set out to investigate whether a cell-free system for the biological synthesis of bimetallic urchin-like nanoparticles is achievable in E. coli. Our team opted to use E. coli because it is a well-documented organism for laboratory purposes, which can grow on relatively simple medium, and has a short doubling time, which makes it easier to culture and scale up to industrial production^34,35. It has also been widely used by several iGEM teams.

We identified three metal-reducing proteins, namely CopA, NapA, and metallothionein, that can reduce silver or gold ions. CopA was deliberately selected from C. metallidurans because it has been identified as an essential player in gold ion reduction within this organism, and is often upregulated upon exposure to gold³⁶. NapA was selected because its role as a silver reductase has been demonstrated in several studies. It has also been identified as a gold reductase; this gene was obtained from C. metallidurans³⁶. Lastly, Metallothionein was selected from C. albicans as it was shown to act as an effective metal reductase to produce homogenous silver nanoparticles³⁷. Metallothionein is a cysteine-rich, metal binding protein capable of reducing metal ions. Therefore, it was worthwhile to investigate whether these metal reductases are capable of reducing both silver and gold metal ions. Altogether, we aimed to use these three metal reducing genes to explore whether the resulting proteins are capable of reducing silver ions (obtained from AgNO₃) to a silver core, followed by reduction of gold ions (obtained from HAuCl₄) to create golden spikes. You can read more about the plasmids we designed on our Parts page.

In addition to using these genes, we also used four other genes from the ASKA collection, which is a set of ORFs cloned from E. coli K12 into a high copy number plasmid PCA24N³⁸. The relevant plasmids were cloned into our E. coli BL21 strain and overexpressed by IPTG induction. The genes selected were NapA, CopA, CueO and MelA. For a detailed explanation of the function of these genes, please visit the ASKA-Effect of adding cell lysate section on our Results page.

Below is an illustration showing our general approach to the biological synthesis of nanoparticle production in a cell-free system (Fig. 4). We used supernatant from a liquid culture of BL21 E. coli strains. This supernatant was supplemented with cell lysate containing CopA, NapA and Metallothionein expressed from the relevant strains. Lastly, the Ag⁺ ions and Au⁺ ions were added to this reaction mixture and left for 24h for nanoparticle production. The produced nanoparticles were then imaged with a Transmission Electron Microscope (TEM). You can find a more detailed description on our Protocols page and Results page.

Why wouldn’t you use chemical synthesis to produce these bimetallic nanoparticles?

Chemical synthesis of nanoparticles largely uses hazardous substances such sodium borohydride, THPC, PVP and hydroxylamine, which are considered to be carcinogenic, cytotoxic, genotoxic, and environmentally harmful^38,39. Furthermore, this may limit the clinical use of nanoparticles due to concerns associated with exposing patients to chemicals that may still be present on the surface of these nanoparticles³⁸. Literature shows that ascorbic acid, a relatively safer and non-toxic chemical, has been used for synthesis of gold urchin-like nanoparticles. However, bimetallic nanoparticles with a silver core and golden spikes have not been obtained using ascorbic acid¹⁸. Additionally, ascorbic acid based synthesis requires specific dilution ratios for suitable nanoparticle production. However, such high dilutions are not feasible at a large industrial scale⁴¹. Therefore, it is critical to develop a sustainable, scalable, and safer method for the production of nanoparticles, which could unlock the full potential of nanotherapeutics. Check out our Entrepreneurship page!

Conclusions and future perspectives

Binanox has contributed to establishing a platform for the biological production of nanoparticles optimal for PTT through synthetic biology; see our Engineering page for more detail. We showed that a microbial system is indeed capable of producing metallic nanoparticles. During the project, we realized that these nanoparticles can also be synthesized to have theranostic properties, where in addition to having a therapeutic effect, the particles may also be used for locating and bioimaging the tumor⁴². We have not been able to fully explore the potential of this idea. However, it is worthwhile to investigate theranostic nanoparticles in this ever-growing field of research! Read more on our Entrepreneurship page for other future business outlooks.