Description

CAPTURE: Combating Pseudomonas aeruginosa Infections with Antimicrobial Peptide Carriers

Antibiotic resistance in Pseudomonas aeruginosa represents a critical challenge in modern healthcare, threatening the efficacy of conventional treatments and patient outcomes. Our project, CAPTURE, addresses this urgent issue with an innovative, multi-faceted approach that combines cutting-edge synthetic biology with advanced drug delivery techniques.

At the heart of our strategy is the power of antimicrobial peptides (AMPs) - nature’s own defense against pathogens. Unlike traditional antibiotics, AMPs disrupt bacterial cell membranes, making it significantly more difficult for bacteria to develop resistance. Our approach goes beyond simply delivering pre-synthesized AMPs. We have engineered a plasmid encoding a potent AMP, placed under the control of a constitutively active, Pseudomonas-specific promoter. This innovative design ensures high levels of peptide synthesis within the target bacteria themselves, bypassing the need for costly external production and purification processes.

To effectively deliver this genetic payload, we employ two distinct delivery systems:

Lipid-based Nanocarriers: Building on their success in applications like mRNA vaccines, our lipid-based nanocarriers offer a versatile platform fine-tuned for optimal encapsulation, stability, and targeting. We enhance their specificity using Gb3 to interact with P. aeruginosa’s LecA protein.
Outer Membrane Vesicles (OMVs): Derived from a specially modified E. coli strain, our engineered OMVs provide a natural and biocompatible delivery vehicle with minimal immunogenicity. We functionalize these OMVs with phage tail proteins via a SpyTag/SpyCatcher system for improved targeting.

What sets CAPTURE apart is the integration of these sophisticated targeting mechanisms. Each delivery system employs a unique approach to enhance specificity and efficacy against P. aeruginosa.

In the following sections, we will explore each component of our project in detail, from the biology of Pseudomonas to our innovative delivery systems and targeting mechanisms. We will also discuss the potential impact of our research on revolutionizing the treatment of antibiotic-resistant P. aeruginosa infections and our vision for future developments in this critical area of healthcare.

P. aeruginosa and P. fluorescens

Distribution of Pseudomonas aeruginosa in Healthcare Settings

Pseudomonas aeruginosa is a remarkably adaptable Gram-negative bacterium that can grow in low-oxygen and aquatic conditions. This is a particular problem in healthcare settings, where P. aeruginosa can colonize and multiply in various water sources.

The rapid multiplication of P. aeruginosa in aquatic systems becomes especially a problem in hospitals. In 2019 alone, this pathogen was responsible for an estimated 51,000 healthcare-associated infections in the United States, highlighting its significant impact on public health.

Due to the widespread development of P. aeruginosa colonies in hospitals, patients and staff come into contact with the hospital germ on a daily basis. Once there, the hospital germ can spread in the water systems, which can infect other patients and healthcare workers can carry the germ from room to room. The germ can spread not only in hygiene facilities, the opportunistic germ P. aeruginosa also finds a place to grow on medical devices or food equipment in hospitals [1].

An example of P. aeruginosa contamination is the Royal Jubilee hospital in Belfast in 2012. The pathogen spread through the hospital water systems, resulting in widespread contamination. Tragically, three infants died because of the insidious pathogen P. aeruginosa [2].

Antibiotic Resistance Mechanisms in Pseudomonas

P. aeruginosa has a considerable repertoire of antibiotic resistance mechanisms (Figure 1):

AMP mechanisms — **Figure 1** Antibiotic Resistance Mechanisms in *P. aeruginosa*

This illustration shows P. aeruginosa’s extremely effective mechanisms for antibiotic resistance:

P. aeruginosa has a reduced membrane permeability, so that the bacterium alters its membrane porins, limiting the entry of antibiotics into the cell.
Efflux pumps actively remove antibiotics from the bacterial cell.
Biofilm can deactivate or modify broad spectrum antibiotics, rendering them ineffective.
Beta-lactamases formation provides an additional layer of protection for bacterial populations exposed to antibiotics.

Biofilm Formation and its Role in Infection Persistence

Biofilm architecture of P. aeruginosa is mostly built by autogenic extracellular polymeric substances and consists of polysaccharides, extracellular DNA (eDNA), proteins and lipids. This matrix accounts for 90% of the biofilm’s biomass, providing P. aeruginosa with an extensive protective shield, which ensures survival and persistence through its mechanisms.

Typical mechanisms are the encapsulation of many bacteria in a film that forms a protective shield around the bacteria. This also favors the development of P. aeruginosa on surfaces and its long-term adhesion. The biofilm guarantees the survival of the bacterium, protecting it from undesirable changes in living conditions such as nutrient decrease and spontaneous temperature changes [3]. In addition, bacteria in biofilms have the ability to escape host immune responses and resist antimicrobial treatments up to 1000 times more than an individual bacteria [4].

Why Pseudomonas fluorescens was Chosen as a Model Organism

P. aeruginosa is a pathogenic bacterium classified as an S2 organism that we could not use in our experiments. To circumvent this obstacle, we searched for a similar organism that we could work with in our lab. The organism of our choice was Pseudomonas fluorescens DSM 50090 - a non-pathogenic strain of the Pseudomonas genus. The relatively close relationship between P. aeruginosa and P. fluorescens, combined with our genomic analysis, was the main rationale for choosing P. fluorescens as our model.

P. fluorescens is a Gram-negative Gamma-proteobacterium of the Pseudomonales order [5]. The bacteria occur primarily in soil and rhizosphere [6], however with some strains also occurring in mammalian hosts [7]. Like P. aeruginosa, it is a biofilm-forming aerobe that is resistant to multiple antibiotics including commonly ampicillin and chloramphenicol [8].

Similarities and Differences to P. aeruginosa

Pseudomonas fluorescens is a low-risk opportunistic pathogen that, similar to P. aeruginosa, can proliferate in aquatic environments [7]. Externally, P. fluorescens appears green due to the production of the pigment pyoverdine [9], while P. aeruginosa exhibits a blue-green color resulting from the combined presence of pyoverdin and pyocyanin.

P. fluorescens exhibits only mild antibiotic resistance and is not considered as dangerous as P. aeruginosa. Additionally, the two species differ in their optimal growth temperatures: P. fluorescens thrives at 25-30°C, while P. aeruginosa is most suited to 37°C, which coincides with human body temperature and contributes to its pathogenicity in humans.

Due to its lower pathogenicity and antibiotic resistance, P. fluorescens is commonly utilized in biotechnology and agricultural research. P. aeruginosa, on the other hand, poses a serious health risk as a pathogen.

How Results with P. fluorescens will translate to P. aeruginosa

Our project faced a significant challenge: while our goal was to target P. aeruginosa, safety precautions prevented us from directly working with this pathogen. To overcome this obstacle, we employed a two-pronged approach combining intensive bioinformatics analysis and comprehensive literature review. Our aim was to identify common expression machinery between P. aeruginosa and its less pathogenic relative, P. fluorescens.

Both species are known biofilm producers, and our analysis uncovered homologous DNA sequences in P. fluorescens for two key P. aeruginosa biofilm-related elements: the pel operon and the FleQ transcription factor. This discovery strongly suggests that similar mechanisms govern biofilm formation in both species.

This finding provides a solid reason for using P. fluorescens as a model organism to test our P. aeruginosa-targeted plasmid design. By utilizing these genetic similarities, we can conduct our experiments safely while still gaining valuable insights applicable to P. aeruginosa.

Our approach demonstrates the power of comparative genomics in overcoming practical limitations in synthetic biology research. It allows us to make meaningful progress towards addressing P. aeruginosa infections without compromising safety.

For detailed information about specific methodologies and results, please refer to our Plasmid Design and Results.

Antimicrobial Peptides (AMPs)

Definition and General Properties

Antimicrobial peptides (AMPs) represent an evolutionarily conserved component of the innate immune defense system [10]. They consist from five to up to one hundred amino acids, most commonly L-forms. They are rich in cationic, positively charged, and hydrophobic amino acids, rendering them amphipathic [11,12,13]. They are capable of killing both Gram-positive [14] and Gram-negative bacteria [15], as well as combating viruses [16] fungal infections [17] and tumor cells [18].

A significant number of AMPs kill bacteria by inducing disruption and leakage of the bacterial membrane and its content. The binding of AMPs to bacterial membranes is typically achieved through peptide-lipid interactions [19]. However, some AMPs use non-lytic mechanisms. These AMPs, which also exhibit antibacterial and antibiofilm activities, can translocate through the membrane and bind to intracellular targets such as DNA, RNA, and components involved in cell wall and protein synthesis [20]. These interactions can ultimately lead to the induction of apoptosis in target cells (see figure 2).

Advantages over Traditional Antibiotics

Antimicrobial resistance (AMR) is reported to be a significant public health concern. It is estimated that in less than 30 years, AMR may become more deadly than cancer, with an estimate of 10 million deaths per year [21]. The misuse of antibiotics in healthcare has led to the emergence of multidrug-resistant (MDR) bacteria, including multidrug-resistant P. aeruginosa (MDR-PA).

Unlike traditional antibiotics with a single target, AMPs can kill pathogens at multiple targets, significantly reducing the emergence of drug-resistant bacteria [22]. Targeting essential, non-protein bacterial structures such as the cytoplasmic membrane and the availability at sites of infection, with cell- and tissue specific production, could have been major obstacles for bacteria to develop highly effective resistance [23].

Another advantage of AMPs is their synergistic effects with antibiotics [24]. A novel therapeutic approach, based on the mechanism of re-sensitization, involves the use of stable peptides at sub-inhibitory concentrations to improve permeability of the bacterial membrane and enable antibiotics to reach its targets with great efficacy [25].

AMPs can also neutralize cellular endotoxins, thus enhancing innate immunity to exert antibacterial effects [26]. P. aeruginosa has natural resistance to a variety of beta-lactam antibiotics and other drugs. Pretreatment with AMPs has been shown to improve the symptoms of pneumonia by recruiting neutrophils and promoting NETosis [27].

Some pathogens, among them P. aeruginosa, showcase a major problem for traditional antibiotics, as the polysaccharide structure on the biofilm will hinder its penetration into the bacteria, reducing the targeting effect of antibiotics [28]. Some AMPs, as mentioned above, have the ability to disrupt the biofilm formation. This can either be by inhibiting the attachment of free cells to the biofilm by altering their morphology, reducing the expression of the primary genes involved in the biofilm formation or degrading the biofilm matrix and polysaccharides [29].

**Figure 3 Schematic diagram of AMPs in combating both Gram-positive and Gram-negative bacteria, fungi, cancer and viruses.** Representing the challenge and the new approach for their clinical application. Adapted from [30].

Challenges in their Clinical Application

The support for the development of AMPs as a solution for AMR, also faces some criticisms [31] :

The assumption that AMPs have a short half-life and are likely to have poor pharmacokinetic properties is based on the peptidic nature of AMPs, as they are susceptible to proteolysis [32]. Given its short half-life its clinical application is limited by its high sensitivity to serum proteases [27]. However, optimizing the structure of AMPs could lead to a breakthrough in this field [33]. Another way to reduce proteolytic degradation could be the usage of nonstandard amino acids in the synthesis of these peptides.
The production of AMPs is expensive [33]. The use of recombinant DNA techniques could facilitate scale-up synthesis, however the peptides are usually lethal to the microorganisms used to produce them.
The use of AMPs as therapeutic agents is hindered by the fact that they do not recognize specific receptors. While AMPs work well with a fully functional immune system, it implies that they cannot be used as a standalone mechanism or have to be optimized for a single function [34].
The therapeutic application of AMPs is hindered by their potential cytotoxicity. The wide variation in cytotoxicity is dependent on the mammalian cell type caused by varying degrees of membrane negative charge [35]. Further tests and optimizations of the AMPs could minimize the damage.

Our project focused on two antimicrobial peptides: Sushi S1 and D-CONGA-Q7.

Sushi S1

Sushi S1 is a naturally occurring AMP derived from the horseshoe crab (Carcinoscorpius rotundicauda). This 34-amino acid peptide exhibits potent antimicrobial activity against Gram-negative bacteria through a multi-step mechanism: binding to bacterial membranes, causing disruption and leakage of cytosolic contents, ultimately leading to bacterial cell death [36]. Sushi S1 demonstrates remarkably low minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) against Gram-negative bacteria including E. coli in general [36] and our target pathogen P. aeruginosa in particular [37].

CONGA-Q7

Our AMP repertoire expanded to D-CONGA-Q7, a synthetic peptide developed by Prof. Wimley’s laboratory at Tulane University School of Medicine. This novel AMP is composed entirely of D-enantiomers of amino acids, which typically renders it resistant to proteolytic degradation. While its precise mechanism of action is still under investigation [38], Prof. Wimley encouraged us to explore the peptide’s effectiveness when expressed directly in the targeted bacteria.

These two AMPs form the foundation of CAPTURE’s antimicrobial strategy. For detailed information on how we specifically utilized Sushi S1 and D-CONGA-Q7 in our project, please refer to our Plasmid Design and Results.

Lipid-based Nanocarriers

CAPTURE introduces an antimicrobial peptide-encoding plasmid directly into P. aeruginosa at the site of infection, allowing for intracellular expression and targeted bacterial killing. To effectively deliver this plasmid into its target bacterium, we explored two distinct delivery systems: lipid-based nanocarriers and outer membrane vesicles (OMVs)(Figure 4).

Classification Transport Vesicles — **Figure 4: Classification of transport vesicles.**

Lipid-based nanocarriers are vesicular structures that can be synthesized in the lab and represent a highly versatile and rapidly advancing technology in the medical field, with applications spanning a wide array of therapeutic areas [39]. They have been instrumental in enhancing delivery of pharmaceutical drugs with poor bioavailability, particularly in cancer treatment, where they improve the targeting and efficacy of chemotherapeutic agents while minimizing side effects [40]. Significant advances have also been made in the field of vaccine delivery, contributing to the development of vaccines for diseases such as hepatitis and influenza [39]. Of particular note is the development of mRNA-based vaccines, including the lipid nanoparticle–formulated SARS-CoV-2 vaccine [41]. Moreover, lipid-based nanocarriers are being explored for the delivery of cutting-edge therapies, including CRISPR-Cas gene-editing systems [42]. These examples highlight just a few of the many innovative applications of lipid-based nanocarriers in modern medicine. In addition to their low immunogenicity, the nanocarriers can be tailored in terms of lipid composition, size, and surface modifications to enhance encapsulation, stability, and target specificity [43].

While there are various categories of lipid-based nanocarriers, our focus is on liposomes and lipid nanoparticles (LNPs) (see Figure 5).

Liposomes, often considered the first generation of nanomedicines [44], have one or more phospholipid bilayers, and form a spherical structure to encapsulate the cargo in an aqueous solution. Liposomes are categorized into subtypes based on size: giant unilamellar vesicles (GUVs, >1000 nm in diameter), large unilamellar vesicles (LUVs, 100–1000 nm), and small unilamellar vesicles (SUVs, <100 nm). LNPs (100 - 400 nm), have a lipid monolayer and their payload is enveloped by reverse micelles. Their interior does not necessarily have to be aqueous. We distinguish between empty LNPs (eLNPs) and plasmid-loaded LNPs (pLNPs). LNPs generally offer greater stability, higher payload capacity, and are more easily scalable in production compared to liposomes [45].

Liposome vs LNP — **Figure 5: Liposome (left) and Lipid Nanoparticle (Right).** Liposomes have one or more lipid bilayers, and form a spherical structure to encapsulate the cargo in an aqueous solution. Lipid Nanoparticles have a lipid monolayer and their payload is enveloped by reverse micelles. Image Created with BioRender.com.

The CAPTURE nanocarriers employ a lipid formulation composed of the lipids POPC, DOTAP and DOPE.

1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) is a phospholipid that belongs to the class of phosphatidylcholines, which are a major component of cell membranes. This is why POPC is commonly used in studies of membrane properties and lipid-ion interactions [46].

1,2-dioleoyl-3-trimethylammonium-propane chloride (DOTAP) is a cationic lipid. Unlike phospholipids, which have a phosphate group as part of their headgroup, cationic lipids feature a positively charged headgroup, such as the quaternary ammonium headgroup of DOTAP. This allows the lipid to form complexes with negatively charged nucleic acids, rendering it effective for gene delivery applications, like for example mRNA vaccines [47].

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) is a helper lipid commonly used in gene therapy in combination with cationic lipids, like DOTAP. DOPE facilitates transfection by promoting the release of the cargo at the target site [48].

chemical structure — **Figure 6: Chemical structures of POPC, DOTAP and DOPE.** Image adapted from avantiresearch.com.

Enhancing Target Specificity

Along with having a lipid composition that ensures high transfection efficiency and minimizes immunogenicity, achieving target specificity is equally crucial for effective lipid-based nanocarriers. One strategy to enhance this specificity is through surface modification of the carrier. In our approach, we focus on exploiting a known interaction between P. aeruginosa and host cells. Research has shown that the interaction between the P. aeruginosa surface lectin Lec-A and the glycosphingolipid globotriaosylceramide (Gb3) on host cell surfaces facilitates bacterial adhesion and uptake [49,50,51]. To leverage this interaction for targeted delivery, our system incorporates Gb3 into the lipid composition of the nanocarrier. This modification is expected to enhance the carriers’ specificity towards P. aeruginosa through the following mechanism:

The Gb3-modified carriers circulate in the system.
When in proximity to P. aeruginosa, the LecA on the bacterial surface recognizes and binds to the Gb3 on our carriers.
This binding increases the likelihood of our carriers interacting with and delivering their payload to P. aeruginosa cells.

By mimicking the natural host-pathogen interaction, we aim to create a more targeted delivery system, potentially improving the efficacy of our antimicrobial approach while minimizing off-target effects.

We invite you to explore the full scope of our work with lipid-based nanocarriers.

Curious about how our lipid nanocarriers performed? On our Results you can explore the extensive work we have done.

In our quest for precision, we developed a novel method to quantify DNA encapsulated in LNPs. This advancement not only enhances our project but also contributes to the broader field of nanocarrier research. Read more about it on our Measurement.

We have created an invaluable resource for the scientific Community – check out our video guide on how to produce and measure the size of lipid-based nanocarriers.

Outer Membrane Vesicles

OMVs are spherical structures, ranging from 20 - 250 nm in diameter, derived from the outer membrane (OM) of Gram-negative bacteria. They typically form through the blebbing of the OM, which occurs due its dissociation from the underlying peptidoglycan (PG) layer [52].

The rate of OMV formation is directly related to the membrane stability, and depends on numerous crosslinks between the PG layer and other proteins. Key components are the porin OmpA, the Tol-Pal complex and Braun’s Lipoprotein (Lpp) [52].

Bacteria utilize OMVs for a variety of purposes, which can be generalized into 4 main categories :

Disposal
Proteinaceous and envelope waste, such as misfolded proteins and PG fragments, are selectively removed from the cell via OMVs
Dialogue
OMVs are involved in both short and long term intercellular communication. For example, they mediate the transport of Pseudomonas Quinolone Signal (PQS), which plays a crucial role in Quorum Sensing in P. aeruginosa [54]. In addition, it has been shown that OMVs play a role in horizontal gene transfer by transporting genes and plasmids [55].
Damage
OMVs export virulence factors [57] that can interact with prokaryotic partners through the encapsulation of peptidoglycan hydrolases [58] and with eukaryotic cells/tissues such as human lung epithelial cells [59].
Defense
Bacteria secrete OMVs to adsorb and neutralize antibiotics and other antimicrobial agents by acting as decoys [60].

Currently, OMVs are being investigated as modular vaccine platforms due to their inability to replicate and their inherent immunogenic properties, such as antigen - or pathogen associated molecular pattern (PAMP) display [62]. OMVs have also been utilized by former iGEM teams, a notable mention being the 2022 team from Xiamen University [63] that used OMVs to deliver endolysins to combat AHPND .

Inspired by their concept, we envisioned a scalable, modular toolbox concept with the potential to target virtually any bacterial pathogen. This involves an easily interchangeable AMP-encoding plasmid encapsulated within OMVs, whose surfaces can be easily modified to achieve specific targeting.

However, not every bacterial strain produces equal amounts of OMVs. Therefore we decided to use the hypervesiculating E. coli omp8 strain kindly provided by Prof. Dr. Daniel Müller (Read more about this on our Human Practices)

The omp8 strain, first characterized in 1998 [64], is a specially engineered variant of BL21(DE3). What makes it special is the absence of the four most abundant β-barrel outer membrane proteins: OmpA, OmpC, OmpF and LamB. These genetic modifications have two significant advantages:

Hypervesiculation: The removal of these proteins weakens the connections between the peptidoglycan layer and the outer membrane, leading to dramatically increased OMV production.
Enhanced protein display: The altered membrane composition allows for greater expression and display of recombinant proteins on the OMV surface [65]. By harnessing the power of this hypervesiculating strain, it is possible to effectively engineer OMVs.

Enhancing Targeting Specificity of OMVs

Our approach to improve the targeting specificity of Outer Membrane Vesicles (OMVs) builds on the work of the 2021 ETH Zürich iGEM Team [66], utilizing a recombinant outer membrane protein: enhanced circularly permuted OmpX (eCPX). Derived from the native E. coli OmpX, eCPX offers the unique advantage of simultaneously displaying peptides on both the N- and C- terminus, without compromising cell viability [67]. To create a modular and versatile targeting system, we integrated eCPX into the SpyTag/SpyCatcher system [68]. The steps to functionalization are:

Expressing a fusion protein on the outer membrane surface, consisting of eCPX and a SpyTag peptide.
Using a SpyCatcher fused to a targeting ligand.
The SpyCatcher “catches” the SpyTag displayed on the OMV surface, effectively functionalizing our OMVs with the targeting ligand.

The eCPX-SpyTag construct was obtained through collaboration with the DiVentura Lab in Freiburg (for more details, please refer to our Human Practices). For our targeting ligands, we selected phage tail proteins due to their inherent specificity and strong binding affinity to bacterial surfaces. Recognizing that antibiotic-resistant bacteria like P. aeruginosa can develop resistance to individual phages, our strategy involves using a phage-tail cocktail. This multi-pronged approach aims to:

Attack the bacteria on multiple fronts, reducing the likelihood of resistance.
Enhance the overall binding efficiency and specificity of our OMVs to P. aeruginosa.

spytag spycatcher — **Figure 9** Illustration showing the modular approach employed with regards to targeting ligand functionalization - OMVs exhibiting eCPX-SpyTag on their surface are bound to targeting ligands consisting of a SpyCatcher fused to a Phage-Tail protein.

The modular nature of our system offers several advantages:

Separation of phage-tail protein production from OMV isolation, allowing for independent optimization of each process.
Scalability, potentially reducing costs for end-users.
Flexibility to “CAPTURE” various bacterial pathogens by simply switching the targeting ligands.

For detailed information on how we specifically utilized eCPX and phage tail proteins in our project, please refer to our Plasmid Design and Results.

Comparing LNPs and OMVs

Our project has been an exciting journey into the world of lipid-based nanocarriers, with the goal of developing an optimal transport platform for our AMP-encoding plasmids. We have focused our efforts on two delivery systems: Lipid Nanoparticles (LNPs) and Outer Membrane Vesicles (OMVs).

Throughout our investigation, we have gained valuable hands-on experience in multiple aspects of nanocarrier design and formation. This practical knowledge has provided us with unique insights into the challenges and opportunities presented by each system.

As we move forward, we are eager to conduct a comprehensive comparison of these two nanocarrier systems.

Aspect	Lipid Nanoparticles (LNPs)	Outer Membrane Vesicles (OMVs)
Composition	Synthetic lipids	Derived from bacterial outer membranes
Adaptability	Highly adaptable, well-researched variations	Modifiable through genetic engineering
Purity	Controlled composition	May contain additional bacterial components, including proteins, lipids, and nucleic acids
Size Control	Highly controllable size distribution through production methods (e.g. extrusion)	Naturally more variable, depending on strain and growth conditions
Encapsulation and Payload Capacity	High encapsulation efficiency with proper formulation of lipids High capacity for various types of cargo (nucleic acids, protein, small molecules)	Encapsulation efficiency for specific components like plasmids can be low. Can be engineered for improved loading
Production and Scalability	Various formation methods with high adaptability Scalable production (e.g. microfluidic mixing) Storage stability may vary, requiring quality controls	Longer production time due to bacterial culture and isolation process Scalable production possible Stable storage at -80°C
Targeting and Specificity	Can be engineered for specific applications Potential for passive accumulation at infection sites Some formulations may have unspecific fusion issues	Natural bacterial components with potential for modification (e.g. SpyCatcher/SpyTag system) Natural fusogenic properties with bacteria
Immunogenicity and Biocompatibility	Highly modular, allowing for minimal immunogenic effects	Natural presence of LPS can induce strong immune responses Immunogenicity can be attenuated through genetic modifications
Biosafety and Regulatory Considerations	Generally safe synthetic materials Some formulations already approved (e.g., mRNA vaccines) GMO regulations apply for plasmid designs	Strict GMO handling requirements Currently in experimental stage with regulatory hurdles Biosafety can be enhanced through genetic engineering
Costs	Higher costs for more specialized designs	Generally lower production costs

Aerosol Delivery System

The Rationale for Choosing Aerosol Delivery

With CAPTURE, we want to develop a treatment that is accessible for as many people as possible. As our lipid-based nanocarriers and OMVs are in solution, aerosol delivery is the most intuitive and effective delivery method. This approach builds upon established practices in pulmonary medicine, where aerosols have successfully delivered various treatments, including siRNA encapsulated in liposomes, directly to the lungs [69].

Our focus on treating P. aeruginosa infections specifically in the lungs makes aerosol delivery particularly promising [70]. By utilizing this well-established method, we are combining proven technology with innovative treatment, setting the foundation for CAPTURE to become a cornerstone in the fight against pulmonary P. aeruginosa infections.

Advantages in Treatment of Lung Infections

A nebulizer-based delivery is particularly suitable for lung infections due to its gentle application. For ventilated patients, CAPTURE can be easily incorporated into their care routine. A simple adapter allows the catheter to be connected directly to the ventilator, enabling treatment during regular ventilation. Additional humidification of the airway can also be added, which then works synergistically with CAPTURE to keep the lungs well humidified and less irritated [71].

By implementing the treatment application directly to the regular ventilation, the plasmid encapsulating transport vesicles are directly delivered to the lungs. This should make the therapy more comfortable for patients than indirectly, intravenous applied medicine.

The delivery of CAPTURE combines ease of use, patient comfort and targeted delivery, establishing a new alternative for the already existing treatments.

Aerosol Deposition and Challenges of Nebulizer Use

Despite the largely established use of nebulizers in the treatment of respiratory conditions, aerosol delivery also presents many challenges that CAPTURE will have to face in the future. The transition from proof-of-concept organisms to the actual treatment of humans requires excessive testing and studies that cannot be achieved in the frameworks of iGEM.

Finding the most effective dosage for aerosol delivery suitable for each health condition, age, weight and gender will take time and careful investigation. To achieve best particle deposition in the lungs airway size, structure and ability of the lung have to be taken into consideration [72]. As animal models offer just little adaptivity to humans, the examination and further development of already established lung models are necessary to investigate the most effective way to administer aerosol delivery of CAPTURE. The use of specific lung models for various parameters like pre-existing health conditions or age would be ideal to test different dosage and treatment conditions [73]. Unfortunately, so far not many lung models are well established and basic research is still lacking.

Another aspect that requires further investigation for the aerosol delivery of CAPTURE is the treatment of children. The deposition of aerosols in the airway of children is not yet fully understood. Furthermore, the application method per se causes difficulties in the therapy for children. One disadvantage that aggravates the treatment of children using nebulizers for ventilation, is the relatively long application time. In comparison to other inhalation devices, the therapy session can be very time consuming [74]. The extended time span can make treatment burdensome for both children and caregivers, as children easily grow impatient and fail to complete the full treatment. Another challenge is that children often struggle to maintain a proper seal around the mouthpiece. This reduces the effective dose of medication inhaled, greatly diminishing the effectiveness of the treatment. Due to these factors, nebulizer use in children is often seen as less efficient and cumbersome [75]. The lengthy delivery times, along with the potential for misuse and added effort, can significantly impact the overall success of the treatment.

Although CAPTURE focuses on the treatment of patients infected with P. aeruginosa at hospitals, the therapy using nebulizers currently rules out self-application of the treatment at home. Compared to other devices like metered-dose inhalers (MDIs) or dry powder inhalers (DPIs), nebulizers are less convenient due to the cumbersome setup, lengthy treatment and risk of misuse [76,74].

While the limitations of aerosol delivery through nebulization pose various challenges that aggravate the treatment of patients, this method allows direct delivery of medication to the infection site without complex inhalation coordination. With advancing studies in lung models and nebulization techniques, CAPTURE is a promising new method to combat multidrug-resistant P. aeruginosa infections.

Potential Impact and Future Directions

Potential Applications beyond P. aeruginosa

Firstly, we want to use CAPTURE to create a new and innovative therapeutic approach against P. aeruginosa that will inhibit the ever-increasing numbers of infections in the future. We are currently on the way to running into an AMR crisis without having any real solutions to the problem. Our current goal is to improve treatment options and to encourage researchers to investigate and search for therapeutic options.

For the future, after the experiments and testing, CAPTURE will give us the opportunity to implement research and technology. With a specific treatment method, CAPTURE will ensure fewer hospital acquired infections and the further weakening of immunocompromised members of our society.

This also means a lower burden for intensive care units and therefore less expenditure. Because if patient safety in hospitals is guaranteed by an effective drug such as CAPTURE and P. aeruginosa cannot multiply, patients are treated more effectively for their original disease, such as cystic fibrosis (CF), and can be discharged earlier.

Retrospectively, this also means that there are fewer P. aeruginosa relapses and the number of infections is sustainably reduced, so that CAPTURE becomes a stable support in the AMR crisis.

Future Steps Towards Clinical Application

For the transition from experimental stages to practical therapeutic use, the first steps in vesicle production would include improving the effectiveness and specificity of CAPTURE. With Gb3 in our lipid composition, there is already a targeting mechanism involved; however, the expansion of lipid components enables further improvement of stability, fusogenicity, and immune tolerance of the delivery vesicle. The lipid composition can be improved by using ionizable lipids instead of the cationic DOTAP, by including PEGylated lipids to form a protective coating for the vesicle and conjugating more advanced targeting ligands like e.g. antibodies to the lipids [77].

In vivo studies, like our first fusion assay with P. aeruginosa, would be expanded to test if the target bacteria are actually transformed with the plasmid, followed by organoid trials and later in vitro studies to test the efficacy but also identify side effects.

The production of vesicles can be upscaled by using microfluidic mixing, a method we have already explored with initial experiments. Optimization of this method can be achieved by finding the optimal flow rate and flow rate ratio [78].

Through these targeted enhancements, we have a highly adaptable and scalable platform for Lipid-based delivery. The careful optimization of lipid composition, alongside improvements in vesicle stability, positions our lipid-based nanocarriers to effectively capture bacterial pathogens while also offering potential for precise plasmid delivery. The modular design ensures that CAPTURE can be fine-tuned to meet specific clinical needs in combating antimicrobial resistance in multiple target organisms.

For our modular approach towards targeting ligand functionalization, we’ve utilized the SpyCatcher/SpyTag system by functionalising OMVs displaying SpyTag with targeting ligands containing a SpyCatcher. Our first course of action would therefore be to switch locations of the two parts, as advised by Dr. Rabe (See our Integrated Human Practices). He suggested pairing the SpyTag with the phage tail protein, as opposed to the SpyCatcher, as fusing larger domains onto the phage tail would possibly adversely influence its functionality. However, plasmid based expression of the eCPX-SpyCatcher construct could run the risk of the same plasmid being encapsulated by the OMV alongside the AMP plasmid. Therefore, we are planning to integrate this construct into the omp8 genome via methods such as CRISPR-Cas or Lambda-Red. These methods can also be used for gene deletions, designed to increase plasmid encapsulation and attenuate lipopolysaccharide-associated endotoxicity (See our Integrated Human Practices Page).

Despite modifications to reduce immunogenicity, proper utilization of our idea would not be possible without the removal of antibiotic resistance markers on the AMP encoding plasmid. Therefore we have planned to integrate a toxin-antitoxin system into our project, wherein the toxin gene would be integrated into the omp8 genome and the produced protein, neutralized by the antitoxin gene on the AMP encoding plasmid. This system eliminates the need for antibiotics within bio-manufacturing processes.

Taken together, these modifications allow us to make a truly scalable, interchangeable, bio-safe modular system for the CAPTURE of bacterial pathogens, development of vaccine platforms, protein delivery and so much more.

References

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