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Understand

Background

There are plenty of patients taking long-term medications to treat chronic conditions. However, It has been estimated that only ~ 50% of patients would adhere to those medications as prescribed. Some patients are tired of the repeated intake of medicine, and some others worry about the side effects of the medicine. In light of these concerns, live biotherapeutic products (LBPs) are being researched as an alternative way to deliver drugs. Some bioactive peptides, or peptide nutraceuticals, can be readily synthesized by the live organism, and potentially treat diseases with fewer side effects.


With the spirit of promoting health from the gut, we constructed an intestinal drug delivery system to stably deliver small peptides in the intestine.


The Urgent Need for Long-Acting Intestinal Drug Delivery

Our inspiration stemmed from the pressing need for long-acting, intestinal-based drug delivery systems. Compared to injectable or intravenous routes, oral and rectal administration methods are easier for patients, more comfortable, non-invasive, and typically lower in cost. They also do not require additional personnel or training for drug administration[1][2]. However, it has been estimated that adherence to chronic oral medications remains low, at around 50%[3]. Long-acting drug delivery systems can reduce dosing frequency and enhance adherence, while also improving pharmacokinetics by maintaining a more stable drug concentration within the therapeutic window[4]. This consistency not only enhances the drug's efficacy and tolerability but also minimizes toxicity, leading to increasing patient adherence.


Discovering an Ideal Material: Peptide Nutraceuticals

Through our research, we identified bioactive peptide nutraceuticals as ideal candidates for promoting health. These compounds possess multiple functions that enhance human health, and their absorption in the intestine is generally more effective than that of single amino acids[5][6]. Prior studies have demonstrated that short peptides can strengthen the epithelial cell barrier, stimulate mucus and promote immune cell differentiation in the submucosa[10]. However, current intestinal delivery systems for small peptides face significant limitations, primarily in the form of capsules, which largely restrict the control over release and absorption.


Engineering LBPs as Delivery Vehicles

As live biotherapeutic products (LBPs) gain broader recognition, the benefits of probiotics in promoting intestinal health, along with advanced genetic tools, facilitate the development of synthetic engineering in this field[7]. Additionally, encapsulation techniques for probiotics are actively being researched, expanding the possibilities for industrialization and clinical trials[8]. In light of the challenges faced by peptide nutraceutical delivery systems, we believe that probiotics are an excellent choice for manufacturing and delivering these substances within the human body.


Attributes of peptides

Different functions and types

Peptide nutraceuticals can be categorized based on their bioactivity into several types, including antioxidant, antihypertensive, anticancer, anti-inflammatory, antimicrobial, antithrombotic, and immunomodulatory peptides[9][10]. These bioactive peptides are ideal nutraceutical candidates due to their diverse health-promoting properties. Although their efficacy may be less than that of specific drugs, they do not typically cause harmful side effects, making them a safer alternative for enhancing health.


Fig. 1 Roles of peptide nutraceuticals.(adapted from Girjia.A.R.2018)


Easy absorption

Intestinal mucosal cells have a unique characteristic: they derive approximately 70% of their nutrients from the intestinal lumen and require direct contact with chyme for proliferation and repair[6]. Small peptides are absorbed intact and are less susceptible to further hydrolysis in the intestine, allowing for efficient uptake into the bloodstream. Once in circulation, these small peptides can directly contribute to tissue protein synthesis, and they can be effectively utilized by organs such as the liver, kidneys, and skin[11].The absorption of small peptides is rapid, energy-efficient, and less prone to saturation of transport carriers. Research indicates that mammals absorb amino acid residues from peptides faster than from free amino acids.


Additionally, by circumventing competition with free amino acids during absorption, small peptides can provide a more balanced intake of amino acids, thereby enhancing protein synthesis efficiency[12].

Improve intestinal health

Previous studies have confirmed that short peptides can strengthen the epithelial cell barrier, increase the production of mucus and antimicrobial proteins, and promote the differentiation of immune cells in the submucosa[10]. Due to the intestinal dysfunction in critically ill patients, the secretion of trypsin is reduced, leading to decreased digestion of whole proteins. Therefore, administering short peptide enteral nutrition preparations, which bypass the step of breaking down whole proteins into short peptides and amino acids, can provide sufficient nutritional substrates for intestinal mucosal cells. This approach is conducive to restoring intestinal barrier function, reducing the risk of intestinal microbiota translocation and enterogenic infections[13] In this way, when we present the peptide on the surface of colonized bacteria in intestine, we can conveniently and continuously release peptide nutraceuticals to boost intestinal health. .


Exemplary example: QEP&AQ

Two representative small peptides, QEP (Gln-Glu-Pro) and AQ (Ala-Gln) have demonstrated significant health benefits when used as therapeutics or nutritional supplements.


QEP(Gln-Glu-Pro)

QEP is shown to mitigate glucose absorption in the intestine, by inhibiting the expression of SGLT1, a glucose cotransporter protein[14].

Fig. 2 Model depicting the presumed RS1-dependent mechanism for downregulation of SGLT1 by QEP at high D-glucose (adapted from Hermann et al. 2020)



In contrast to traditional SGLT1 inhibitors, the small peptide has additional antidiabetic effects such as increasing insulin sensitivity and normalizing elevating fasting glucose. Detailed comparisons betweuen QEP and traditional SGLT1 inhibitors are shown in the table below.

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AQ(Ala-Gln)

AQ belongs to the family of glutamine supplement. After entering the body, it will be digested into alanine and glutamine. Compared to glutamine, it has increased solublility, stablility, and ease of absorbtion[16].

Fig. 3 Mechanisms of enteral and parenteral glutamine (GLN) supply.(Cruzat V et al.2018)


As the most abundant free amino acid in the bloodstream, glutamine exhibits important functions. Some of them are listed below.

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Various delivery methods have been applied to deliver these prospective small peptides to the target site. However, current delivery systems are not satisfying.

Part 4: Current delivery system

Current delivery systems include controlled-release microparticle depots, in-situ-forming polymer matrix, implants, etc[23]. Although some of them have already had clinical products launched in the market, they still possess shortcomings like invasive injection, unsustainable and non-inducible release.

Invasive injection:

Delivery methods like intravenous and subcutaneous routes require injection, which is invasive and brings pain and suffering to patients.

Unsustainable release:

Traditional delivery methods like oral administration are unsustainable. Patients have to take medicine frequently (for example, 3 times a day), which increases their burden.

Non-inducible release:

Delivery systems like controlled-release microparticle depots release therapeutics slowly for up to 6 months[24]. However, drugs are released constitutively and not due to the microenvironment response, making the system more uncontrollable.

Fig. 4 Disadvantages of current small peptide delivery systems


Part 5: Our solution

To create a non-invasive, sustainable, and controllable delivery system, we genetically engineer Escherichia coli into probiotic Intestide. Our proposed design encompasses three interconnected modules—Secretion, Adhesion, and QS (Quorum Sensing) safety—and involves two distinct strains of engineered bacteria: Peptide Producers and Controllers.

Fig. 5 Genetic circuit of Intestide


Secretion module

The Secretion module is developed for the peptide producer strain, facilitating the secretion of small peptides. This module features a recombinant transmembrane protein, Lpp'OmpA, which would be.integrated into the outer membrane of E. coli upong syntheses. By synthesizing a chimera protein consisting of this small peptide and transmembrane protein, we enabled their delivery into the surrounding environment[25].

Fig. 6 Schematic view of the delivery system


Adhesion module

This module would be incorporated into both the peptide Producer and Controller strains, which consists of Binding between HSP60 and LAP. HSP60, also present as a membrane protein on intestinal cells, interacts with LAP through a β-barrel protein structure displayed on the outer membrane of E. coli. This arrangement results in the formation of a strain-layered matrix that securely adheres to the intestinal lining[26][27].

Fig. 7 The inspiration for the Adhesion module. HSP60, as a membrane protein on intestinal cells, can bind to LAP(blue dots on bacteria) present in the outer membrane of E. coli (adapted from Tsinghua iGEM 2021)


Fig. 8 The expected effect for the Adhesion module, which adheres to the engineering bacteria and intestine (adapted from Timmis, K. et. al 2021)


Biosafety module

The Biosafety module comprises two distinct components within the peptide producer and controller strains. Based on a reciprocal activation principle, whereby one strain's product induces the activation of the other's gene promoter, this module regulates the population dynamics of the peptide producer and controller strains to ensure optimal small peptide production[28]. Furthermore, the effective functioning of the QS-safety mechanism is contingent upon the cohabitation of both bacterial strains and the formation of the adhesion matrix. The viability of these strains is primarily sustained within the low-oxygen environment of the intestine, effectively mitigating the risk of bacterial leakage[29][30].

Fig. 9 Shcematic view of QS system


To sum up, our engineered E. coli will be delivered into the intestine, in which they will colonize and reproduce with the aid of the adhesion module. The secretion module displays small peptides on the surface of bacteria, which are cleaved by enterokinase in the intestine and conduct their tasks. To confirm the biosafety, peptide producer and controller regulate the number of each other through the QS system.

Fig. 10 Schematic view of Intestide


References


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