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Understand
  • Part 1: Our application cycle
  • Engineering probiotics EcN
  • Engineering probiotics are encapsulated in the chosen material
  • Engineering probiotics orally administrate into the patient intestine
  • Adhesion to form biofilm and start safety system
  • Secretion-tailored peptide into the intestine under or not under induction
  • Replenish bacteria regularly
  • Part 2: Comparison
  • Comparison with current methods
  • Our design
  • References

    • Part 1: Our application cycle

    Engineering probiotics EcN

    Escherichia coli Nissle 1917 (EcN) is considered as probiotic for its health benefits such as anti-inflammatory effects, inhibition of pathogenic bacteria[1], toddlers-tolerated and infants-tolerated[2]. Nowadays, the genetic tools of EcN are well established as plasmids pMUT1/2 system are fully invented. The outcomes of EcN also gain a significant number of novel therapies for immunological and metabolic disorders. For example, engineered EcN senses tetrathionate and utilizes it for microcin H47 production, which helps in salmonella inhibition to treat infection[3]. You can refer to A white paper of Engineering probiotics EcN get more information.



    ●View E.coli Nissle 1917 White Paper


    In this way, we aim to build our system in EcN as the final product.



    Engineering probiotics are encapsulated in the chosen material

    1.Delivery of probiotics

    ● Encapsulation materials are important

    Improve the intestinal delivery efficiency of the probiotics due to the adverse effect of the harsh gut environment and increase colonization rate

    ● The disadvantage of the current delivery of probiotics and our solution

    a.Disadvantage:
    Currently, probiotics in clinical trials or commercial products are mostly freeze-dried powders or encapsulated in oral capsules. However, these methods just provide simple protective effects for probiotics. Some of it may result in early release of probiotics after oral administration, reducing the bioavailability of these probiotics[4].
    b.Our solution:
    Therefore, our probiotics are engineered to express Listeria adhesion protein (LAP) from a nonpathogenic Listeria (Listeria innocua) which mediates the bacterial attachment to the intestinal mucus layer contributing to the intestinal colonization.


    2.What kind of encapsulation materials we can use?

    ● Natural materials

    a.Polysaccharides
    Polysaccharides are commonly used encapsulation materials for probiotics. It is low cost, biocompatibility, accessibility, and pH-responsive property.
    b.Chitosan
    It is also a promising coating material for probiotics due to its biocompatible, mucoadhesive, film-forming, and modifiable properties. In addition, chitosan can be metabolized by the action of enzymes produced by gut microorganisms like lysozyme. However, it can dissolve in the acidic environment and cause unexpected release. It is more useful to enhance the strength of capsules.

    ● Synthetic materials

    a.pH-responsive material
    A conventional approach to target the colon is pH-dependent polymers coating on capsules/ tablets/ nano-micro carriers that protect the drugs in the upper gastric tract and deliver the drugs to different segments upon degradation by fluid pH.



    Engineering probiotics orally administrate into the patient intestine

    1.Use oral administration

    ● Oral administration is more comfortable, non-invasive, and typically lower in cost compared to injection.


    2.Gastrointestinal environment

    ● Our target colonization zone is in the intestine. For further consideration of a more practical application, we need to take a number of factors in the intestine into account: absorptive area, pH, cells and transporters, permeability, and bacterial population, are both a challenge and an opportunity for targeted drug delivery[5][6].



    Adhesion to form biofilm and start safety system

    1.LAP and HSP60 adhesion have a bonus benefit

    ● It can also promote the probiotics' competition with other intestinal pathogenic bacteria, fight against gut infections of pathogenic Listeria, and promote delivery efficiency as well as the possibility to contribute to the positive effect in clinical result[7].



    Secretion-tailored peptide into the intestine under or not under induction

    1.Short peptides have several functional types

    ● Peptide nutraceuticals can be categorized based on their bioactivity into several types, including antioxidant, antihypertensive, anticancer, anti-inflammatory, antimicrobial, antithrombotic, and immunomodulatory peptides[8].

    ● The doctor can choose to use different functional peptides to assist in promoting health according to the medical examination report.


    2.Choose induction or not induction

    ● The promoter before the Lpp'OmpA-FLAG-peptide-GST can be changed to adapt to different conditions. For example, if the patient only needs to decrease the glucose absorption to maintain blood sugar when glucose concentration in the intestines grows to a certain level, a promoter called Trz-Omp[9] can be placed in the circuit to sense the level change. In this way, when the intestinal glucose concentration rises to the level, the QEP (peptide that can decrease glucose absorption) can be secreted to the membrane of the bacteria.

    Fig. 1 The schematic view of Trz-OmpR system(Jan T. Panteli et al., 2016)
    Fig. 2 Initial design of genetic circuit of sensing and secretion module



    Replenish bacteria regularly

    As the probiotics has the ability to colonize and proliferate in the intestine, we supposed the cycle of replenishing is much longer than taking peptides orally everyday cycle.

    Part 2: The comparison of our application with current long-time delivery technology

    Comparison with current innovative methods to deliver peptide

    1.The in-situ-forming polymer matrix[10][11]

    ● It uses a gel or semi-solid tissue at the injection site.Although the matrix slowly releases the drug over a prolonged period of time, the drug degradation in solution form is a difficult problem.


    2.The non-invasive delivery systems[12]

    ● It can use oral, transdermal, inhalation, and mucosal delivery to get the aim of unpainful injection. However, the disadvantages lie in slow absorption, low surface area for absorption, and harsh chemical environment, which all limit the possibility of the strategy.


    3.The controlled-release microparticle depots[13]

    ● It can release the therapeutic drugs for up to 6 months, lowering the needle frequently. However, the strategy faces physical and chemical mechanisms of instability that act on the peptides and proteins and cannot adjust the release dose to the change of chemicals in the body.



    Our design(pros&cons)

    1. The disadvantage:

    ● Unpredictable bacteria growth may happen under different human health conditions and intestinal conditions

    ● The tailored promoter and peptide may be quite a cost

    ● The engineering probiotics for nutrition replenishment may be unacceptable in traditional views


    2. The advantage

    ● Wide audience:

    This approach is particularly beneficial for specific populations: elderly individuals experiencing digestive decline, athletes needing nitrogen supplementation without increased gastrointestinal strain, and those with poor digestion or nutritional deficiencies[14]. For these groups, supplementing amino acids in the form of small peptides can significantly improve amino acid absorption and fulfill the body's nitrogen and amino acid requirements.

    ● Great innovation:

    Engineering probiotics with safety and innovation

    ● Long-time administration

    References


    [1]

    Sonnenborn, U. Escherichia coli strain Nissle 1917—from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiology Letters 363, fnw212 (2016).

    [2]

    Henker, J. et al. The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhoea in infants and toddlers. Eur J Pediatr 166, 311-318 (2007).

    [3]

    Palmer, J. D. et al. Engineered Probiotic for the Inhibition of Salmonella via Tetrathionate-Induced Production of Microcin H47. ACS Infect. Dis. 4, 39-45 (2018).

    [4]

    Li, C., Wang, Z.-X., Xiao, H. & Wu, F.-G. Intestinal Delivery of Probiotics: Materials, Strategies, and Applications. Advanced Materials 36, 2310174 (2024).

    [6]

    Chu, J. N. & Traverso, G. Foundations of gastrointestinal-based drug delivery and future developments. Nat Rev Gastroenterol Hepatol 19, 219-238 (2022).

    [7]

    Drolia, R. et al. Receptor-targeted engineered probiotics mitigate lethal Listeria infection. Nat Commun 11, 6344 (2020).


    [8]

    Girija, A. R. Peptide nutraceuticals. in Peptide Applications in Biomedicine, Biotechnology and Bioengineering 157-181 (Elsevier, 2018). doi:10.1016/B978-0-08-100736-5.00006-5.

    [9]

    Panteli, J. T. & Forbes, N. S. Engineered bacteria detect spatial profiles in glucose concentration within solid tumor cell masses. Biotech & Bioengineering 113, 2474-2484 (2016).

    [10]

    Vargason, A. M., Anselmo, A. C. & Mitragotri, S. The evolution of commercial drug delivery technologies. Nat Biomed Eng 5, 951-967 (2021).

    [11]

    Madan, M., Bajaj, A., Lewis, S., Udupa, N. & Baig, J. A. In Situ Forming Polymeric Drug Delivery Systems. Indian J Pharm Sci 71, 242-251 (2009).

    [12]

    Anselmo, A. C., Gokarn, Y. & Mitragotri, S. Non-invasive delivery strategies for biologics. Nat Rev Drug Discov 18, 19-40 (2019).

    [13]

    Schwendeman, S. P., Shah, R. B., Bailey, B. A. & Schwendeman, A. S. Injectable controlled release depots for large molecules. Journal of Controlled Release 190, 240-253 (2014).

    [14]

    Bröer, S. Intestinal Amino Acid Transport and Metabolic Health. Annual Review of Nutrition 43, 73-99 (2023).