Team:Thessaly/Implementation
Implementation Overview
Overview
Here we present the method in which the capsule shall be implemented in the real world, along with the related safety aspects and challenges that come with it. The capsule is intended to be consumed by people diagnosed with Crohn’s Disease or Ulcerative Colitis, with few exceptions, which are presented in the user manual of the capsule.
Safety
Due to various parameters the interaction between the capsule, the genetically engineered
bacteria and human cells faces many safety, security, and ethical risks.
The first barrier that protects us and the environment is the capsule itself that acts as a
mechanical barrier. Furthermore, we designed, as a second barrier, a “kill switch” to ensure
that we can keep the patients safe.
Retention of the capsule is the most significant complication associated with such devices.
According to studies, capsule retention occurred at a 1.4% rate. Retention is related
exclusively to capsule size and the risk of it is minimized significantly for capsules 28mm in
length and 12mm in diameter. The best approach is using smaller materials within the
capsule. This becomes a difficult task when it comes to batteries, as progress to increase
their energy-density ratio is much slower compared to most electronics. However, recent
advances in material sciences with fully biodegradable batteries, and edible printable
electronics can decrease the risk factors ingestible capsules are associated with. Recently
developed hydrogel devices may be another solution to overcome capsule retention. To
become the new norm in patient care, scientists and engineers specializing in fields of
electronics and material sciences should focus on the safety and economic factor.
Another major concern was selecting the transmission frequency for our data. We selected
the ISM (433 - 434.8MHz) commercial communications band, because it presents effective
transmission and acceptable tissue penetration in regard to safety, compared to most
commercially available frequency bands.
Our proposed “kill switch” circuit is designed with the help of iGEM Ohio State University
and it is based on the mazEF toxin-antitoxin system, an “addiction module”, composed of
two genes, mazF, toxin gene & mazE, gene producing the antitoxin product. We have placed
the mazF under the control of two inducible promoters, the ParaBAD, an arabinose-induced
promoter and a cold-induced one stemming from the cold shock CspA gene.
In case the capsule leaks, resulting in the bacteria being released inside the patient’s body,
exogenous consumption of arabinose shall trigger the activation of the mazF gene, leading
to the bacterias’ imminent death. When the capsule is excreted naturally from the human
body, the temperature difference will turn the switch on killing the bacteria and
safeguarding the environment.
Some objections to our product were expected due to the misinformation and the aversion
towards trying new methods to solve problems. What can we do to change that mindset? Of
course, the answer lies in the education and public engagement department.
Advantages compared to current methods
Treatment in IBD
To combat this issue, a range of drugs, natural products, and treatment options have been employed by physicians, even though the multi-dimensionality of the disease and uncertainty of the severity of the disease present challenges (Seyedian et al., 2019). Generally, medications are subcategorized into two groups, traditional small molecule drugs, and biopharmaceuticals. The first class includes common anti-inflammatory and immunosuppressants drugs, like aminosalicylates, thiopurines, and steroids that can provide symptom improvement but do not change the overall disease course in inflammatory bowel diseases, while they are often accompanied with side reactions, such as susceptibility to infections (Balestrieri et al., 2020) (Na & Moon, 2019). On the other hand, elucidation of the immunopathology of IBDs has led to the development of targeted therapies and has unlocked a new era in IBD treatment embedded in the second class, which includes monoclonal antibodies and related agents (Lee et al., 2018). In fact, the development of biologics has exhibited better overall management of the disease, including lower rates of surgery and better long- term clinical and patient-reported outcomes (Weisshof et al., 2018). However, it has been reported that the nature of the protein is coupled with some drawbacks, like the need for parental administration, sterile cell culture for the production environment, and immunogenicity followed by host-immune counter-reactions. Side effects might also appear as well (Na & Moon, 2019) (Al Mijan & Lim, 2018).
Alternative IBD treatment options have led to the use of effective natural-based products (Day et al., 2019). The so-called “nutraceuticals”, comprise any food-derived natural products, like bioactive peptides or fatty acids, that appear to have health-promoting features Additionally, recent research provides evidence that many nutrients and food elements can cure IBD symptoms (Al Mijan & Lim, 2018). This health-promoting effect is likely based on the fact that the human body interacts with and depends on the microbiome, a wide range of probiotic bacteria that have established a mutually beneficial symbiotic relationship, as they perform some very vital roles regarding the host’s health. One category of these bacteria is one of the lactic acid bacteria, that are key organisms in the production of vitamin B12, which cannot be synthesized by either animals, plants, or fungi. The products of probiotics and their energy source, prebiotics, belong to the “nutraceutical” umbrella (Thursby et al., 2017).
Probiotics encompass a broad category of bacteria including their metabolic byproducts, that promote well-being, especially gut health and intestinal homeostasis. (Ahn et al., 2020) (Plaza-Diaz et al., 2019) (Day et al., 2019) (Penner et al., 2005) (Larussa et al., 2017). They produce biomolecules, such as lactic acid, bacteriocins, and Reactive Oxygen Species (ROS) that assist the immune system in fighting pathogenic organisms. Moreover, they supply humans with vitamin K, vitamin B12, pyridoxine, biotin, folate, etc., they metabolize or deactivate compounds with toxicodynamic potential and elicit useful immunostimulatory roles, both pro-and anti-inflammatory (Day et al., 2019). This has proved to be useful in managing plenty of digestive disorders such as acute, nosocomial, and antibiotic-associated diarrhea and Clostridium difficile–associated diarrhea and some inflammatory bowel disorders in adults (Plaza-Diaz et al., 2019).
Despite these health-promoting effects, our project revolves around the probiotics’ ability to produce Short Chain Fatty Acids (SCFAs) (Markowiak-Kopeć et al., 2020). SCFAs are a category of carboxylic acids (acetic, propionic, and butyric acid) that exert important immunoregulatory and physiological roles. These metabolites are produced by intestinal bacterial fermentation of luminal carbohydrates and proteins. Enterocytes depend on short-chain fatty acids as their primary energy source, thus strengthening the intestinal gut barrier (Parada et al., 2018). They contribute to intestinal health, by lowering the pH level in the colon, thus limiting the growth of pathogens. Furthermore, they have signaling capacities, as they can bind to GPCR receptors embedded in the intestinal membrane and initiate immunoregulatory signaling pathways that maintain immunological homeostasis in the intestines, hence their role as potential anti-inflammatory compounds has been investigated (Venegas et al., 2019), (Russo et al., 2019). They act upon B cells inducing them to secrete sIgA. They act as epigenetic immunoregulators by inhibiting histone deacetylases (HDAC) in neutrophils, monocytes, and dendritic cells, thereby inactivating nuclear factor-κB and downregulating tumor necrosis factor (TNF), a process that promotes an anti-inflammatory cellular mechanism. They can also bind to and initiate the activation of GPCR receptors embedded in enterocytes and immune cells leading to the expression of anti-inflammatory cytokines, maintaining immune homeostasis (Rooks & Garrett, 2016).
DIAGNOSIS IN IBD
IBD diagnosis and treatment are complex, as the disease’s etiology and pathophysiology still are not fully understood. To complicate matters further, IBD can manifest in organs other than the gut. One such organ is the oral cavity. For IBD diagnosis, many methods have been used, including both the use of digital devices and biomarkers. A clinically important biomarker is calprotectin, a protein that is associated with inflammatory diseases (Manceau et al., 2017).
The hitherto most established clinical use of calprotectin is fecal determination, where its levels correlate to neutrophil migration through the gut wall and thus reflect inflammation in IBD (Ayling et al., 2018). Consequently, fecal calprotectin has proven to be useful in the diagnostic work-up of patients with suspected IBD, but can also be used as a surrogate marker for the presence of endoscopic inflammation, as well as an early prognostic marker for upcoming disease activity in patients in remission, thus holding the potential to monitor the disease over time.
A different approach to this matter is the use of the potential of salivary calprotectin to reflect disease activity and treatment response using serum concentrations as a positive control. Investigating effects on salivary calprotectin was found that calprotectin in the saliva is elevated in IBD patients and is related to IBD activity and treatment (Majster M, et. al., 2019).
As they normally are a byproduct of the intestinal microbiome metabolism, any alteration or depletion of the gut microbiota lead to lower concentrations of SCFAs, making them potential biomarkers. One aspect of their nature is that their concentration reflects intestinal homeostasis and well-being and may prove useful in evaluating the intestinal microbiome, providing a useful insight regarding a differential diagnosis for gut-related diseases, like IBDs. While, another aspect is that these molecules, through the variety of mechanisms by which they exhibit their immunoregulatory activity, can provide relief from IBD-related symptoms (Parada et al., 2019).
The human gastrointestinal (GI) tract remains largely inaccessible to modern researchers. The diagnosis of IBDs has proven to be a real challenge as it rests on a multidisciplinary workup, based on clinical evaluation integrated with a combination of endoscopic, histological, radiological, and/or biochemical investigations (Tontini et al., 2015). Traditional methods for diagnosis include endoscopy, colonoscopy, laparoscopy, and open surgery. All of the aforementioned methods suggest an invasive way of diagnosis, as their execution is only possible through the usage of various devices that not only provide an expensive method of IBD detection but also reduce the patient’s quality of life submitting them to uncomfortable and strenuous circumstances. This has led to late diagnosis and treatment of GI diseases (Spray et al., 2001). A non-invasive and safe approach to reach GI tract is the use of ingestible sensors.
The first ingestible devices were introduced in the late ’50s (JACOBSON et al., 1957). Since then, breakthroughs in semiconductor microelectronics have led to sophisticated ingestible sensors for evaluation of the GI tract using optical images, gases sensors, pH, and temperature monitoring (Van der Schaar et al., 2013) ( Hafezi et al., 2015) (Iddan et al., 2000). According to “Smart Pills technologies Market 2012-2017” the global smart pills market has reached 1 billion USD by 2017. Small intestines’ length is on average 6 m, with a 2.5-3 cm in diameter (Kourosh et al., 2017), thus, to be swallowable the capsule should not exceed 28 mm in length and 12 mm in diameter. The proposed capsule has dimensions of 21mm x 12 mm (length x diameter) (Mimee et al., 2018). To support the electric circuit, silver oxide batteries are used. While, Li-ion batteries seem to present much higher power density, compared to silver oxide batteries, they are not preferred due to the health issues if they are exposed to the GI environment (Kourosh et al., 2017).
Amalthea is a complete, personalized, modular platform, which provides full monitoring of the GI tract’s health by accomplishing diagnosis, evaluation of the gut microbiota, and treatment of IBD in a non-invasive way. Compared to current methods both in the field of diagnosis and treatment, Amalthea advantages each approach.
Challenges
Many obstacles may occur in the prosecution of this project. For example, with regard to
heterologous expression of high molecular weight eukaryotic proteins, this poses as a
challenge, as it is difficult to achieve and is often accompanied with various troubleshooting,
like its inability to survive in high temperatures, even if progress has been marked (Gialama
et al., 2017). Although, it is restricted to certain temperatures, thus making it impossible to
survive inside the human body, this obstacle could be overcome, due to the insulating
material of the capsule.
In a parallel manner, other challenges that might be faced concern the structure of the
capsule. Firstly, we want to make the capsule more commercial and accessible to as many
people as possible. In order to do that, we should focus on making it more affordable and
patient centered. This is achieved by carefully selecting materials that are cheaper but
evenly effective to meet the requirements of our project’s design. Customer experience is
greatly improved with a user manual for the capsule, a minimized diagnosis time, direct
readout of the data on the user’s phone and by letting the user keep their eating habits as is.
Implementation
The capsule will be available for distribution from pharmacies, contained within a box with a user manual to accompany it. It must be prescribed by a doctor. The patient will have the option to ingest it with the presence of a physician, or in the comfort of their own home, though it is recommended that a physician be present for the process. As it is described in detail in the user manual, the patient should have the receiver antenna plugged to their phone and keep it in the specified distance for the data to be transferred effectively. Also, the user is instructed to keep a safe distance from devices that use EM waves, so that they do not interfere with the data transmission and corrupt the data. After the capsule has completed all its measurements, it will be naturally excreted from the body, after which it is advised to pick it up and dispense it in an electronics recycling bin. Further detailed instructions are introduced in the user manual that comes with the capsule.
Envision
The bio-electronic information generated will be instantly stored to the cloud on the user’s smartphone or computer, for convenient readout. It will be accessible, at any given time, to patients and to professionals, who will use it to evaluate the microbiome and visualize the functionality of the intestinal flora. This information is then used to provide a personalized treatment based on the needs of each patient, to achieve relief of symptoms and to design a daily diet without dietary restrictions.
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