| NU_Kazakhstan - iGEM 2023

Summary

Our main goal was to develop a system that would be highly specific and would have high efficiency without damaging the healthy tissues. Specifically, we worked on developing an innovative, localized, and controlled anti-cancer drug release system against solid tumors by using engineered bacteria embedded in a hydrogel. The main advantage of using hydrogel is the minimization of adverse events and side effects of the treatment on normal cells. Hydrogel-based bacterial therapeutics is a highly controlled, smart, and localized treatment option.

What is hydrogel?

Hydrogel is a polymeric 3D mesh-like network capable of dissolving high quantities of water [1]. The reagents used for hydrogel synthesis are usually hydrophilic molecules, which form cross-links with each other and, instead of dissolving, swell in water [2]. Due to this and other physical properties, such as porosity, softness, and high water content, hydrogels have a wide range of potential applications in tissue engineering, regenerative medicine, biosensors, drug delivery, and other biomedical settings [3]. Moreover, the gels exhibit biocompatibility and biodegradability, which makes their incorporation into the human body safe and lacking side effects [4]. In therapeutic delivery, hydrogels offer many advantages. Most importantly, spatial and temporal control of drug release can be maintained by adjustment of the hydrogel pore size. Additionally, their compositional tuning can lead to the development of hydrogels responsive to temperature, pH, and other external factor changes.

Why do we need to encapsulate bacteria?

Bacterial therapy has been an active research area over the last decades. However, despite all of the potential, this method does not usually get to clinical trials because introducing bacterial agents into the body can lead to severe side effects, up to sepsis development [5]. The leading causes of these adverse events are an uncontrolled spread of foreign organisms and toxic molecule production by bacterial cells. To minimize dangerous side effects and make the treatment safe, we used endotoxin-free E. coli cells and encapsulated our bacterial therapeutics into a hydrogel [6].

Since pores of the hydrogel are small enough to restrain bacterial migration to the outside environments, encapsulating E. coli cells into the gel prevents their unwanted proliferation in the organism, retaining them in the injection site specifically. Therefore, such systems introduce high-level control over the therapeutic agent. It is essential to note that the pore size of our hydrogel is also large enough to permit metabolite exchange between bacterial cells in the surrounding environment. Therefore, due to such chemical permeability, encapsulation of bacteria does not inhibit their proliferation and metabolic activities. Along with this, other properties of hydrogels make them closely resemble the extracellular matrix, which is why these scaffolds are excellent for storing bioactive therapeutics [7].

Another advantage of our system is that trapping E. coli cells inside the hydrogel makes bacteria “hidden” from the immune cells [8]. This significantly reduces the destruction of therapeutic agents by host cells and, as a result, increases potential drug payload to the cancer site.

Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel

Figure 1. Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel structure

The CellCare system comprises genetically modified E. coli cells encapsulated into Chitosan-based hydrogel modified by the addition of β-glycerophosphate for temperature sensitivity and Hyaluronic acid to adjust the pore size. Chitosan is a linear polysaccharide made of β-(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine. Chitosan is produced from the chitin shells of marine animals by treating the surfaces with alkaline substances. The hydrogel's components are biocompatible, biodegradable, minimally toxic, and FDA-approved. For instance, chitosan degradation by nonspecific enzymes (lysozyme mainly) leads to the formation of essential polysaccharides, which can be incorporated into the organism's metabolic pathways or excreted naturally [9].

The addition of β-glycerophosphate turns the chitosan solution into a temperature-sensitive hydrogel, solidifying at body temperature. The gel is liquid at room temperature. In this form, bacteria or other therapeutic agents can be easily trapped inside it [10]. After temperature increases, the mixture undergoes sol-gel transition, and monolithic hydrogel gets formed. The mechanism behind physical state change is as follows: Mixing of β-glycerophosphate and chitosan leads to electrostatic interaction of the amino groups of both reagents [11]. As temperature rises, these noncovalent bonds are destroyed, and the viscosity of chitosan sharply increases due to its deacetylation, which leads to hydrogen bonding between hydrophobic and hydrophilic interchain components. Aside from that, electrostatic forces between chitosan and β-glycerophosphate through the ammonium and the phosphate groups also play a role in the gelation [10]. After cooling, chitosan aggregation is reduced due to weakening of hydrophobic interactions. On the other hand, hydrogen bonding becomes more prevalent. As a result, hydrogel exhibits partial thermal reversibility. The degradation rate of the gel depends on the degree of deacetylation of chitosan [10]. We used the reagent with a high degree of deacetylation. Hydrogels with this type of chitosan have a long residence period (about three weeks). Moreover, no detectable inflammation was observed after such hydrogel injection into a rat model. These hydrogels have a neutral pH of 7.15.

Through various tests, we could determine that Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel does not significantly affect bacterial cells and allows them to be metabolically active and increase rapidly. This treatment would not require frequent administration because of the prolonged period required for gel degradation.

As mentioned, hyaluronic acid (linear polymer made of repeating β-1,4-d-glucuronic acid-β-1,3-N-acetyl-d-glucosamine disaccharide units) was primarily added to the hydrogel to reduce the pore size. However, aside from minimizing bacterial leakage from the scaffold, hyaluronic acid improves many of the hydrogel properties. First, Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel exhibits increased adhesion to cancer cells [12]. This is because hyaluronic acid is a primary ligand for the CD44 receptor, the expression of which is elevated in many tumor types. Hence, CH/GP/HA hydrogel will demonstrate better sticking to cancer cells and, as a result, the chances of gel (and encapsulated bacteria) migration are significantly reduced. Aside from that, the addition of hyaluronic acid increases the mechanical strength of the hydrogel. CH/GP/HA hydrogel also exhibits pH-dependent drug release: lower pH values lead to elevated rates of macromolecule diffusion. These two properties enhance the specificity of the treatment to tumor cells.

Administration route and temperature sensitivity

Initially, hydrogels were synthesized in vitro and then surgically implanted into a patient’s body. However, over the recent decades, non-invasive application methods have been developed. In situ-forming hydrogels are heteropolymers that form cross-linkings upon injection into the target site. Such innovations are highly beneficial since they minimize the invasivity of therapeutics delivery as in situ forming hydrogels can be injected with the help of standard needles.

Initially, Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel synthesized in our lab has a liquid form. It undergoes sol-gel transition upon exposure to body temperatures: hydrogel fully solidifies at 36.62 °C. Since the Chitosan/β-glycerophosphate/Hyaluronic acid mixture produces an in situ forming hydrogel, our system can be directly administered into the tumor site (intratumoral injection). Such an administration route is used for drug delivery of other therapeutics for solid carcinoma (such as breast cancer) treatment.

Administration procedure consists of the following steps:
1. Synthesis of liquid hydrogel and encapsulation of genetically modified E. coli cells that produce Colicin E1
2. Intratumoral injection of hydrogel
3. Sol-gel transition and solidification of hydrogel at 36.62 °C
4. Localized effect of the therapy on cancer cells

Figure 2. Simplified steps of CellCare system preparation and injection