Project Description

“Antimicrobials play a crucial role in preventing and treating infections and diseases in humans, animals and plants, but their overuse and misuse is the main driver of antimicrobial resistance (AMR). AMR is a multi-faceted global challenge known as a “silent pandemic”. It is considered one of the top ten global public health threats to humanity in the 21st century.” (WHO) ^[1].
The World Health Organization (WHO) has been raising alarm about a "silent pandemic" since the fall of 2021. Unlike the sudden and widespread outbreak of coronavirus, antibiotic resistance develops insidiously and unnoticed, but poses an equally global threat to all. The urgency of this issue is already apparent, with an estimated 1.3 million people dying each year due to bacterial antimicrobial resistance. If no action is taken, this number could continue to rise dramatically ^[2]. For the first time in ages, we must acknowledge that not all bacterial infections are treatable. This highlights the seriousness of a global problem whose unchecked consequences will impact the very foundations of the modern world ^[3].

Our transFERRITIN project steps in at this point. With the growing problem of antibiotic- and therapy-resistant germs, we believe it is important to find alternatives to standard antibiotic therapy. Unfortunately, the development of new antibiotics is slow, difficult, and expensive, and investments of pharmaceutical companies decrease. This is partly because new antibiotics are initially held back as a reserve, making it less profitable for companies to invest in them^[4]. Our project aims to find a solution to antibiotic resistance by exploring alternative ideas and developing new therapies.

Our project seeks to approach the problem from more than one point:

1. The amount of prescribed antibiotics has to be reduced to a minimum. By developing targeted delivery system with increased efficiency we will reduce the needed quantity of application. Thus, fewer antibiotics will come in circulation preventing new resistances to emerge.
2. Common antibiotics affect many bacteria in the body, not just the pathogenic. In consequence, the body’s microbiome gets harmed, leading to the patient suffering from long-term consequences. The specificity of transFERRITIN aims to overcome this limitation by sparing out bacteria of the microbiome and specifically targeting pathogens only. This is an advantage over broad-spectrum antibiotics, which can negatively affect the diversity of the gut microbiome ^[5].
3. Through the synergistic effects of the plant-based, antimicrobial components (see “antimicrobial components” below), we aim to develop an effective strategy against resistant bacteria. The presence of various components at once creates a more challenging environment for bacteria as several random mutations that result in resistance would be required simultaneously. If that's not the case, the bacteria cannot survive and any potential resistance cannot be passed on.

This presents an advantage over conventional antibiotic therapy, and we anticipate that transFERRITIN will provide an alternative solution to counteract the growing problem of antibiotic resistance.

Building transFERRITIN

With the implementation of a modular drug delivery system, our idea is to design a “trojan horse” that can introduce a selection of different synergistically acting substances into targeted bacteria strains. This way, we want to test a therapeutic method to transport antimicrobial substances directly into the targeted cell. The drug delivery system will be designed based on modification of the protein complex ferritin, which is found in humans, animals and even archaea ^[7]. This protein is essential for the storage of iron and due to its storage capacity, the protein complex is characterized by a cavity in which chemical substances can be trapped, stored, and transported ^[8]. We have attached cell-penetrating peptides (CPPs) onto the outer surface of ferritin. CPPs are short peptides that can easily cross a cell membrane and carry cargo along with them ^[9],[10]. Our aim is for the CPP-loaded ferritin to enter the bacterial cell. As ferritin is a major iron supplier, this system represents a "trojan horse" for bacteria. Once CPP-Ferritin enters the cell, the trapped components will be released, killing the bacterial cell from the inside.

Our drug delivery system could potentially target both harmful pathogens and beneficial bacteria in our microbiota because CPPs are not specific to single bacteria strains. To achieve specificity of our transport system, we have attached nanobodies - smaller versions of antibiodies - to the surface of ferritin using click chemistry. These nanobodies are designed for binding a specific surface molecule of a pathogen. Furthermore, the use of click chemistry allows for the quick exchange of nanobodies, making it easier to target different bacteria.

Our aim is to maintain modularity in our transport system implementation. This entails the creation of a toolbox that can be utilized to construct a versatile transport system capable of penetrating different bacteria, depending on the composition of the nanobodies, CPPs, and the encapsulated active ingredients. Our ultimate goal is to apply this transport system to various infections, as per the WHO's 2017 list of 12 prioritized resistant pathogens ^[5]. In our iGEM project, we have focused on Escherichia coli and aimed to achieve a proof of concept. After a successful implementation, our focus will shift to Pseudomonas aeruginosa, a carbapenem-resistant germ classified under priority 1. ^[5]

Hereby we introduce you to the components making up the brilliant system of transFERRITIN!

What Is Ferritin?

The foundation of our project: ferritin, a globular and hollow protein complex consisting of 24 monomers. It is found in almost all living organisms, including animals, plants, bacteria and fungi, serving a function as an iron storage. With a diameter of 12 nm and an interior cavity measuring 8 nm, ferritin can store up to 4500 iron atoms (fig. 1). Storing of iron is a crucial component of cellular iron homeostasis, preventing toxic effects of iron excess. Ferritin comprises of two subunits: heavy (21 kDA) and light (19 kDA) chains, with varying proportions in different tissues. The heavy chain contains a catalytic region that oxidizes Fe(II) to Fe(III), rendering it insoluble causing nucleation at its core. ^[11]

Each ferritin subunit consists of a four-α-helix bundle (A, B, C & D) along with a short fifth E-α-helix at the C-terminus which reaches from the outside to the inside of the container. Another feature is a loop (L) of 19 residues that connects the C-terminus of the B-α-Helix to the N-terminus of the C-α-Helix (fig. 2). This arrangement results in the N-terminus, L-loop, and the A- and C-α-helices being accessible on the surface, while the C-terminus, and B- and D-α-helices form the interior cavity. ^[12]

Ferritin subunits exhibit self-assembly with consistent size, structure and monodispersity. Moreover, they display remarkable thermal and pH stability, tolerating temperatures of 80 - 100 °C and pH levels between 3 - 10. Further shifting of pH into its extremes disassembles ferritin into its monomers, which is reversible by restoring favorable conditions (fig 3).^[11]

These characteristics make ferritin highly interesting in the context of medical application. The fact that ferritin is a protein familiar to the human body, makes it biocompatible and degradable. Therefore, ferritin is a promising candidate for drug delivery platforms. Substances can be conjugated to its surface, encapsulated, or enter through its pores. Additionally, ferritin structure is susceptible to a variety of modifications allowing inclusion of various molecules to add new functions and characteristics. Ferritin is already under research for applications in anti-tumor therapies and vaccinations. ^[11]

We see in its characteristics and structure a platform that is highly modular, capable of serving multiple purposes and with plenty of research data, it is the perfect base for our project!

Our goal, finding a way to treat antibiotic resistant bacteria had its first component: the nanocage.

Drug Loading

To combat antibiotic resistant bacteria, we intend to find a drug that can substitute antibiotics and load it into the nanocage. As mentioned earlier, ferritin allows different methods of drug loading. The cargo’s characteristics, as well as the intended application are factors that must be taken into account when settling for one of the methods below.

1. Channel diffusion

   Ferritins surface has six hydrophobic and eight hydrophilic three-fold channels suitable for passive loading of small molecules. The cavity of ferritin has a strong negative charge that allows positively charged ions to easily pass through its pores to accumulate at its core, which is its main function as an iron storage. ^[9]

2. Dis- & reassembly

   As described earlier, ferritin can be disassembled under extreme pH values below 3 or above 10, with reassembly occurring at neutral conditions ^[11]. Fat-soluble natural active compounds have been successfully encapsulated by using this method. However, too extreme a pH can completely disassociate the ferritin, causing aggregation and insoluble precipitates. Additionally, pH-sensitive molecules are challenging to encapsulate ^[9].

3. Thermal expansion

   Ferritin features a four-fold channel that can be expanded to over 1 nm by thermal fluctuations. This method can achieve higher loading efficiency, recovery rates and stability than pH dependent loading, though longer incubation times are required ^[9].

4. Chemical coupling

   Protein surfaces rarely include amino acids such as cysteine and lysine, yet they are found on ferritin’s surface ^[12]. Cysteine's sulfhydryl (-SH) and lysine’s amine (NH₂) group allow chemical conjugations with drugs ^{[12, 13]}. Furthermore, the C-termini (-OH) also can be used for chemical coupling ^[12].

For our antimicrobial substances we have selected four components: Quercitine, Flavone, Rutin and PHB Ester, all components of the plant extract of Sofoxin. More details on these can be found in our Antimicrobial components section.

Chemical conjugation has the disadvantage that the active ingredients have to be chemically modified in order to be equipped with the corresponding functional groups. Since we want to include several substances, the (cost) effort would be too high. Moreover, we are not sure whether bacteria would be able to release the active substance by breaking the covalent bond. See the section from the with Prof. Dr. Tobias Beck for more details.^[10]

Diffusion through channels is limited by the pore size, requires the transfer of the active ingredient into e.g. a Cu(II) salt and performs worse than the other methods. ^[10]

Therefore, we have concluded that the pH-dependent diss- & reassembly is the most suitable for our intentions.

Protein Modifications

Ferritin’s structure provides three distinct interfaces that can be modified. With modifications we can introduce new abilities, functions and characteristics that will assist us in our mission.

1. Surface modifications

   Modified surfaces can introduce targeting capabilitys, increase half life, or trigger specific cellular responses. Common modifications include peptides, anti- and nanobodies, antigens, functional proteins and as previously mentioned, drugs. ^[12]

2. Intersubunit modifications

   Modifying intersubunits could influence the dis- and reassembly mechanism. However, we were told by Prof. Dr. Tobias Beck that the fusion of peptides typically does not have any impact on assembly. Additionally, they could alter the pH-values needed for the dis- and reassembly mechanism. Another possibility is designing metal binding dependent assembly mechanisms. ^[12]

3. Interior modifications

   Altering the interior might be necessary if the cargo loading of distinct molecules proves to be challenging. It can be modified to bind molecules or to change the cavity’s charge. For larger cargo, the interior space can be increased by removing parts of the C-terminus ^[15].

Bioengineering

The modification of ferritin can be achieved through a number of methods, each with varying levels of complexity, benefits, downsides and usage. The choice of bioengineering approach depends on the modification and its intended purpose.

1. Genetic fusion

   Genetic fusion can achieve structural stability ^[12] . However, genetic fusions must take structure and functionality in consideration, further explained below.

2. Chemical coupling

   Chemical coupling, previously described for drug loading, is also suitable for coupling the attachment of antibodies or peptides. It may result in heterogenous products. ^[9]

3. Chemically inducible dimerization (CID)

   CID employs small molecules to induce irreversible binding of two different proteins. It is comparable to a bifunctional crosslinker. It has high affinity, specifics and kinetics but is restricted to the N- and C-terminus. ^[12]

4. Click chemistry

   Click chemistry referred to as fast, selective, and high yield reactions between functional groups. It allows conjugation of proteins at nearly any site, though it requires incorporation of non-canonical amino acids and functionalization of the proteins of interest. ^[12]

5. Enzyme-catalyzed conjugation

   This method uses a catalysis to activate residues on amino acids, allowing irreversible binding to a second protein. It offers greater flexibility than genetic fusion or CID, as it is not restricted to the N- or C-terminus.^[12]

6. Tag/Catcher Technology

   The Tag/Catcher technology is based on extracellular bacterial proteins and offers a way to create covalent bonds between proteins. These are split into two fragments and modified rationally to create tag and catcher, that can spontaneously bind. These, however, are restricted to the N- and C-termini.^[12]

The Surface of our construct was modified by two distinct methods:

CPPs have been introduced to the surface by genetic fusion. It proved to be the most suitable for our intended purposes and available time. The structure and function of the CPPs required its N-terminus to be unconfined and flexible as well as stably bound to ferritin for penetration of the complete construct.

The Nanobodies are introduced by click reaction. By introducing the functional group, consisting of an amber-codon our construct will be highly modular. The amber-codon was integrated into the L-Loop of ferritin.

Genetics

When modifying proteins, genetic makeup and structure play an essential role. Proteins contain varying levels of conservation in their genetic makeup that, if disturbed, can result in structural and functional changes. While the N- and C-termini usually only pose steric challenges, modifications in other areas need to be carefully planned.

Below is a ferritin subunit that is colorized in a spectrum from red to blue, indicating low conservation (blue) to highly conserved (red) (fig. 4). ^[16] White indicates that the data is insufficient for conclusions, however the E-helix (white) has been partially removed in other studies without consequences. ^[15]

In figure 5, various alterations of ferritin are depicted to show their localization and steric effects. In the top row the N-terminus, in the middle the L-loop and in the bottom the C-terminus have been modified. Steric effects could impact assembly or expression if not taken into consideration ^[5].

The data visualized here suggest our construct’s structure with our CPPs conjugated to the N-terminus and the nanobodies to the L-Loop.

Alternative Nanocages

We briefly considered using encapsulin as our nanocage. Its functions and characteristics are similar. A strikingly attractive difference is its cavity’s size, ranging from 24 to 42 nm. However, encapsulin is of bacterial origin, as prokaryotes lack compartmentalization, encapsulin carries out this function. A Protein with bacterial origin will most likely trigger an immune response which is why we have decided to use ferritin instead. ^[18]

Our goal with transFERRITIN is the establishment of a modular drug delivery system that can efficiently traverse the impermeable phospholipid bilayer of bacterial cell membranes, facilitating intracellular access and release an antimicrobial agent directly into the cells. Components responsible for facilitating the entry of our carrier into pathogenic bacteria are cell-penetrating peptides (CPPs).^[19]

Considering this, we inserted one CPP at the N-terminus of the ferritin subunit of our container (fig. 6). Due to their ability to transport high molecular weight polar molecules across membranes, they have been also called Trojan horses .^[20] The Trojan horse refers to the mythical tale of a stratagem used by the ancient Greeks to enter the city of Troy and win the war against their historic enemies.^[21] In drug delivery strategies, the metaphor of a Trojan horse is employed to securely access a target located within cells by utilizing, for example, CPPs as a protective guise to penetrate the pathogenic cell membranes, thereby carrying the bioactive compound.

Why CPPs?

Cell-penetrating peptides (CPPs) are short peptides with the capability of crossing biological membranes and entering cells. These peptides usually consist of 5 to 42 amino acids and are being researched for their potential in drug delivery and biotechnology.

Cell-penetrating peptides can be used to transport a variety of molecules, such as proteins, into cells. Their ability to pass cell membranes makes them possible valuable tools in medical applications.

Even though the sequences for the CPPs we used were already added to the iGEM registry, we gave them a new purpose in the competition: being part of a modular drug delivery system.

Choosing the Right CPPs

CPPs encompass three principal categories: cationic, anionic and amphipathic CPPs. Among these, cationic CPPs exhibit superior penetration efficiency in E. coli DH5α with low cytotoxicity.^[22] Given this observation, we decided to use the following three CPPs, all of which were deemed suitable according to both the generally applicable safety guidelines: R9 (BBa_K4669001), R12 (BBa_K4669002) and TAT (BBa_K1202006).

Expression of the CPP-Ferritin Construct

Our goal was to use CPPs to transport the ferritin container inside the cells. Based on a suggestion from Dr. Dirk Becker, we have decided against covalently binding the CPPs to the previously expressed ferritin. Instead, our goal is to co-express ferritin and the CPPs in E. coli. To connect the CPPs to ferritin, a glycine-serine linker is used (fig. 8). This linker guarantees minimal steric hindrances while ensuring the utmost flexibility of the CPPs during attachment to the protein.^[24] The pET22b(+)-plasmid contains the protein sequence for ferritin (BBa_K4669000) followed by the glycine-serine linker (GGGGS) (BBa_K4669003) that connects the ferritin with the CPPs R9 (BBa_K4669004) / R12 (BBa_K4669006) / TAT (BBa_K4669005) (fig. 9-10). The C-terminus of the CPPs is fused to the N-terminus of the ferritin. It was necessary for the N-terminus to be used, as the CPPs must still function using their N-terminus.

Open Questions:

• How do the CPPs interact with the human body?
  • Dr. Clemens Wülfing told us that TAT peptides naturally occur in HI viruses. These viruses are known to be able to hide from the immune system so we assume the CPP would not cause an immune response. However, we don’t know how the other two CPPs we chose interact with the human body.

• Are there non-immunogenic alternatives for CPPs that can be fused to our ferritin container? (For an outlook you can read the interview with Dr. Clemens Wülfing)

• Is there a difference between the penetration of competent bacterial cells and the penetration of “normal” pathogenic cells with CPPs?
  • What about differences between the penetration of Gram-negative and Gram-positive bacteria?

However, CPPs are not cell specific. To achieve specificity we needed another part for our modular drug delivery system: nanobodies

Specificity plays an important role in our project. It is what makes our approach stand out from other commonly known applications of antibiotics or antimicrobial substances. By providing specificity, we can establish a system for targeted delivery. Thus, we can transport substances specifically to their place of action, improving their effectiveness and in addition protecting the body’s own microbiome.

transFERRITIN is capable of being specific because of one of its components: nanobodies. We couple these smaller versions of antibodies to the surface of our ferritin.

Coupling The Nanobody

Our investigation of the structure above showed that the L-loop of ferritin is barely conserved (fig. 11). In addition, when looking at the assembled container, it is visible that the L-loops of two subunits are always next to each other in the assembly and they face outwards (fig. 12) ^[25]. In total, this conformation is found twelve times all over the ferritin, providing twelve possible binding sites. This made the L-loop a perfect target for us to mutate some amino acids to provide a binding site for the nanobody.

Bispecific Nanobodies

Our first idea was to design a bispecific nanobody and to fuse two binding sites of different nanobodies together. One part would specifically bind to the shape of the interface of two neighboring L-loops, allowing the binding of twelve nanobodies to the outer surface of ferritin (fig. 13). The other side would bind to our target structure of the pathogen.

The fusion of both nanobody domains would happen on the genetic level. Thus, the bispecific construct could have been expressed, purified and then added to the mixture in vitro. When targeting a new pathogen, the fusion on the genetic level would have to be redone for the new combination. However, for the application later on, the ferritin construct and the nanobodies could be mixed as wished for the needed application.

With this idea we went straight into the talk with Dr. Alejandro Rojas-Fernandez, an expert for nanobodies.

Click Chemistry - The New Approach

After talking to Dr. Alejandro Rojas-Fernandez, we decided to develop a new approach for coupling the nanobody to the surface. He liked the idea, but gave us the advice to try a new method: we now introduce a non-canonical amino acid (ncAA) into the ferritin backbone which will be modified for click chemistry. Just like we will modify the nanobody for the click chemistry reaction (mov. 1). The great advantage of using this approach: We can precisely control where the nanobody will bind to the ferritin surface due to its ability to only click to the ncAA-introduced points.

Identifying the coupling site

To introduce the ncAA we have to mutate the backbone of the ferritin to integrate the Amber codon (TAG/UAG). This codon is one of the three common stop codons and would normally cause the termination of translation. To make use of it for our purposes, we have to introduce a second plasmid to E. coli that contains a pair of a fitting tRNA and the aminoacyl-tRNA synthetase (aaRS). The tRNA has the corresponding anticodon for binding to the amber codon and can be loaded with a ncAA by the aaRS. Thus, by finding a fitting pair for the chosen ncAA, it can be integrated into the ferritin backbone without stopping its expression.

We had a look at two factors for finding the codon to mutate for constructing the coupling site:

1. Conservation of the amino acids in the L-loop
2. Position of the residues in the L-loop

We decided to mutate the lysine at position 88 located in the L-loop (fig. 14-15). It faces outward and when assembled, the residues of two neighboring subunits are not interfering with each other (fig. 16-17). We introduce the mutation K88TAG per QuikChange mutagenesis and obtain the mutated parts:

Finding a fitting ncAA and its counterpart

Using the database iNClusive of iGEM Freiburg 2022 we found the ncAA trans-cyclooct-2-ene-lysine (TCO-lysine) (fig. 18). It is an amino acid most similar to lysine, which contributes to avoiding deviant folding of the ferritin. It is applicable for click chemistry and works by using stop codon suppression TAG (Amber).^[26]

A fitting counterpart that could be used in a click chemistry is tetrazine (Tz) (fig. 19).

They react in a rapid reaction and form a conjugate for covalent binding (fig.20). Tetrazine also reacts with amino groups (NH2) of proteins and thus will bind covalent to the nanobody.

Designing the helper plasmid

The tRNA and aaRS used for introducing TCO-lysine are the Mm-tRNA Pyl CUA (BBa_K172204, from now on referred to as Pyl-tRNA) and the Mm-PylRS (BBa_K4669011, from now on referred to as PylRS), both coming from Methanosarcina mazei^[27].

We used a backbone having a different origin of replication, another system for induction and a different antibiotic resistance than our expression plasmid for ferritin to avoid complications. We added the PylRS and two copies of the Pyl-tRNA. By using araBAD promoters, our system is inducible by adding arabinose (fig. 21). With that we established the helper plasmid.

Choosing A Nanobody

What nanobody to use entirely depends on which pathogen to target. The advantage of how we built our system is that it is modular. Thus, we can easily change the nanobody as soon as we want to target something else. For a proof of concept of showing that the coupling works, we are working with an antiGFP nanobody. Later, we plan to use various nanobodies that will vary from targeting specific lipopolysaccharides to cover multiple organisms, to targeting markers that are specific to one pathogen.

Getting all the parts together

Ferritin + TCO

After we co-transformed an expression strain of E. coli with both the ferritin-Amber expression plasmid and the helper plasmid, we have to add TCO-lysine to the medium of the expression culture. The bacteria will take up the ncAA and thus will be able to express the ferritin including the TCO-lysine.

Nanobody + Tetrazine

For modifying the antiGFP nanobody with tetrazine, we used Me-Tet-PEG2-NHS (SiChem, SC-8821). In a PEGylation reaction, the NHS-ester will bind to an amino group of the antiGFP nanobody (fig. 22).

Ferritin + Nanobody

After preparing the modified ferritin and nanobody separately, they can simply be incubated together. The click chemistry reaction is supposed to happen fast and reliable (fig. 23).

At the end we have the completed transFERRITIN complex, ready for encapsulating substances and for application.

Open questions:

We are currently in the phase of expressing the modified ferritin and unfortunately did not have the time to do experiments with the completed construct. What we would have to examine later on are the following points:

1. How strong will the expression rate of the ferritin in E. coli will be affected due to using ncAAs?
2. Are alternatives, like pathogen recognition receptors or complement receptors, which we discussed with Dr. Clemens Wülfing, more fitting?

Apart from creating the transport system, we must also determine the active ingredients to be transported in the ferritin container. Our transport system is modular and can swap components based on the target or usage. Therefore, the cargo should also be adjustable as required. We chose plant-based antimicrobial substances instead of antibiotics for our project. You can learn about Tobias Beck or above under Ferritin.

Choosing something different than antibiotics

It is important to identify and explore new compounds against bacterial infections, beyond conventional antibiotics, for several reasons:

• Antibiotic resistance is a global health crisis. Overuse and misuse of antibiotics have led to the emergence of antibiotic-resistant bacteria strains. The pace of antibiotic resistance surpasses the development of new antibiotics. New active substances are needed to explore new treatment strategies.
• Some bacterial infections are resistant to multiple classes of antibiotics, making them difficult to treat. New compounds can use different mechanisms to combat these multi-resistant strains.
• The development of new antibiotics is both time-consuming and labour-intensive. Furthermore, it is not very profitable for pharmaceutical companies since newly approved antibiotics are initially held back as reserve antibiotics, making it difficult for them to make a profit.
• Herbal components can work together to create a synergistic effect resulting in broader effectiveness compared to a single antibiotic.
• The combined effects of multiple components increase the difficulty of bacteria developing resistance. This is due to the requirement for bacteria to mutate randomly at the same time to develop resistance to all components. Without successfully doing so, the bacteria cannot survive and the potential for resistance cannot be transmitted.^[1,4,28]

Sofoxin as an antimicrobial plant extract

Sofoxin is a natural remedy created by SjF Hanse Scientific GmbH that is made from plant extracts found in the a powerful antimicrobial effect against a variety of bacterial germs, but the exact mechanism is currently unknown. What has been discovered is that the individual components of the plant extract work synergistically to achieve a stronger effect when combined. This makes it more difficult for bacteria to develop resistance because they would have to form a random mutation against multiple components at once. To create an effective alternative to antibiotic therapy, we've chosen to combine several plant components and encapsulate them in a ferritin container. For more information, check out our Human Practices interview with Dr. Juri Smirnov and SjF Hanse Scientific GmbH.^[28]

Selection of single components from the extract

Although the plant extract has proven to be effective against various bacteria, we have decided to work with only four specific components from the extract. We learned through discussions with three experts in the field - Dr. Juri Smirnov Prof. Dr. Wolfgang Streit and Dr. Thomas Grunwald - that the varying composition of plant extracts from batch to batch makes it difficult to guarantee a standardized cargo for our ferritin. For the sake of quality management, quality assurance, and safety, it is necessary to standardize and control the composition and concentration of each individual component. For human application, each ferritin container must contain the same cargo. Therefore, following the recommendation of the team at SjF Hanse Scientific GmbH, we have narrowed down the plant extract to four components that best align with our project idea, taking into account their solubility, size, and potency: quercetin, rutin, flavone, and parabenes. We want to test each of these components individually and in combinations to determine their synergistic effects.

Selected components

In recent years, natural compounds have attracted increasing interest for their potential antimicrobial properties. Among these compounds, quercetin, rutin, flavone and parabenes have attracted attention due to their broad-spectrum antimicrobial activity.

• Quercetin is a flavonoid found in many fruits, vegetables, and grains. In addition to its antioxidant properties, it has been extensively studied for its antimicrobial properties as well. It has demonstrated antibacterial activity against various bacterial strains, such as E. coli . It exerts its antimicrobial effect by disrupting the bacterial cell membrane and interfering with bacterial DNA replication.

  

• Rutin is another flavonoid commonly found in various plants and fruits while it is primarily known for its antioxidant properties, rutin also exhibits antimicrobial potential. Rutin has shown antibacterial effects against pathogens like Streptococcus mutans, a bacterium associated with dental caries. It may help in preventing oral infections.

  

• Flavone belongs to a subgroup of flavonoids, which are secondary plant compounds found in various plants such as citrus fruits, celery and broccoli. Its antibacterial potential is due to e.g. disruption of the cell membrane or inhibition of enzymes.

  

• PHB esters (p-Hydroxybenzoic acid esters), also known as parabens, are chemical compounds that occur naturally but can also be produced synthetically. In nature, parabens are found in various fruits such as blueberries, carrots, but also herbs. However, the paraben content is usually quite low. It is assumed that they are produced by the plant to defend itself against microorganisms. The antibacterial effect of parabens is based on the fact that they can inhibit enzyme activities. Therefore, they are already used as preservatives in the food and cosmetics industry.^[29,30]

  

Our hypothesis

Our aim with transFERRITIN is to create a specialized drug delivery system that can effectively infiltrate bacterial pathogens and directly release an antimicrobial agent into the cells. Here's what we're trying to accomplish:

• It is believed that transporting herbal components or antibiotics inside a cell could lead to more effective results. This is because if the active ingredient's mechanism of action occurs inside the cell, such as binding to proteins and inhibiting function, a lower dose can achieve the same effect by transporting it directly to the site of action. This method can reduce the loss of the active ingredient during transportation, resulting in a more cost-effective and resource-efficient process with a lower concentration of the ingredient.
• If the antimicrobial components derived from plants have a greater impact on bacteria growth when used within the cell rather than externally, transFERRITIN transport could be a promising substitute for traditional antibiotic therapy. This alternative could be used by doctors to treat patients, reducing the need for antibiotics and preventing the development of bacterial resistance caused by overuse. Our approach can help combat the emergence of new resistance.
• It is currently unclear how the plant components quercetin, rutin, flavone and PHB ester function within cells. However, based on tests conducted by SjF Hanse Scientific GmbH, it has been demonstrated that the combination of these components is effective against resistant germs like MRSA (methicillin-resistant S. aureus) and VRE (vancomycin-resistant Enterococcus), suggesting that the pathogens resistance mechanisms do not impact the components effectiveness. It is possible that these components could even inhibit a pathogen's resistance mechanism, reactivating antibiotics that were previously ineffective against resistant pathogens. To achieve this, a combination of plant components and antibiotics would be used, with the plant components inhibiting the bacterium's resistance mechanism and allowing the antibiotic to work effectively once again.

Open questions

The start-up SjF Hanse Scientific GmbH sponsored the components that needed to be encapsulated. Throughout the project, we communicated closely with Dr. Juri Smirnov and the team at Hanse Scientific. When selecting the components, we relied on Hanse Scientific's recommendation, which was based on their extensive research on efficacy, synergies among the components, and solubility. However, after receiving the components, we conducted laboratory tests to confirm the results on inhibiting bacterial growth. Due to time constraints, we were unable to test the encapsulation of the components in the ferritin container, but we plan to do so in the future after the iGEM competition.

• Can we guarantee to encapsulate always the same amount and composition of components?
• Do the selected components work with every pathogen?
• Do they possibly support the effect of antibiotics or reactivate their effect on resistant bacteria?
• Are they more effective if we can transport them into the bacterial cell instead of using them from the outside?