| NU_Kazakhstan - iGEM 2023

Summary

Throughout our project, we synthesized temperature-sensitive in situ forming Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel.

Solidification of the hydrogel occurs at 36.62 °C.

Pore size depends directly on the amount of hyaluronic acid used during the preparation. The pore size of Chitosan/β-glycerophosphate hydrogel is about 56.61 ± 18.82 μm. In 0.5% HA hydrogel, the average pore size equals 18.39 μm, while 11.54 μm pores were observed when 1% HA was added. Pores do not form when 3% or more HA is used. In the hydrogel chosen for our system, 2% HA is added, and the average pore size is about 3.94 μm.

The hydrogel can efficiently retain bacterial cells after encapsulation without allowing them to migrate freely to the outside environments. It does not have a severe negative effect on bacterial growth and enables their proliferation.

In Vitro Erosion Test results indicate that the hydrogel can be sustained in the injection site for over 21 days.

Due to unexpected logistics problems, we could not conduct bacterial transformation and further analysis as the plasmid still needed to be delivered to our team. However, we conducted virtual simulations of agarose gel electrophoresis and SDS-PAGE gel electrophoresis to determine the expected results of these experiments.

After the plasmid arrives, we will check its performance in the lab according to our experimental design.

Hydrogel synthesis and basic sol-gel transition evaluation

One of the key desired properties of the hydrogel for our system is temperature sensitivity. After several trials with different compositions, we synthesized an in situ forming a hydrogel. As demonstrated in Figure 1, the initial formulation (chitosan/glycerol) did not exhibit temperature sensitivity as the gels remained liquid after prolonged exposure to body temperature. As a result, we had to continue synthesis with another composition. Figures 2-4 illustrate the physical state of Chitosan/β-glycerophosphate hydrogel. Initially, the gel appeared to be a viscous liquid (Figure 2). After putting the hydrogel into the water bath at 37°C for 8 min, the gel became more dense and less transparent. Holding the gel in a water bath for 10 min resulted in the complete solidification of the hydrogel (Figure 3). Afterward, the gel was placed on the bench (room temperature ~25°C). The gel slowly returned to the liquid form after 40 min in these conditions. Figure 4 demonstrates the physical state of the gel after 40 min on the bench. Therefore, the Chitosan/β-glycerophosphate mixture produces an in situ forming temperature-sensitive hydrogel.

Figure 1. Chitosan/Glycerol hydrogels after 20 min in the water bath at 37°C

Figure 2. Chitosan/β-glycerophosphate hydrogel right after the synthesis

Figure 3. Chitosan/β-glycerophosphate hydrogel after 10 min in the water bath at 37°C

Figure 4. Chitosan/β-glycerophosphate hydrogel after 40 min on the bench (25°C)

For pore size adjustment, hyaluronic acid was added to the mixture. Figures 5-6 demonstrate the physical states of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel. Right after the synthesis, the gel could freely flow upon tube inversion (Figure 5). Subsequently, the hydrogel was placed into the incubator set to 37°C. As can be observed in Figure 6, the sample became completely solid after 8 min. Rheometric analysis must be performed for a more precise determination of gelation time and temperature.

Figure 5. Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel right after the synthesis

Figure 6. Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel
after 8 min in the static incubator at 37°C

Rheological measurements

Rheological measurements were conducted to make precise conclusions regarding the solidification temperature and to evaluate how hydrogel deforms in response to applied forces (flow behavior). The storage modulus describes the elastic properties of the hydrogel, while the viscosity properties are characterized by the mechanical loss modulus [3]. The temperature sweep test with Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel solution between 5 and 50 °C showed a sol–gel transition behavior at 36.62 °C (Figure 7). When the temperature increased to 50 °C, the elastic modulus increased to 43.913 Pa. Both the storage modulus (G′) and loss modulus (G″) decreased slightly from 8.7 °C to almost 30.2 °C. Then, the storage modulus began to increase gradually, confirming the hydrogel's thermosensitive behavior [4]. The intersection point of G' and G" (tan δ: G"/G' = 1) represents the hydrogel solution's incipient gel point or the sol-gel transition. At a moment of 36.62 °C, it gets solidified. For practical purposes, a liquid is typically considered viscous if tan > 100:1 = 100, whereas a solid is considered ideally elastic if tan 1:100 = 0.01.

Steady shear flow (flow curves) report the dependency of the viscosity of the hydrogel on the applied shear rate [5]. This characteristic of hydrogels is advantageous for therapeutics delivery, tissue engineering, and other biomedical applications. The viscosity of the hydrogel stood at 88895E+05 mPA*s and then started to decline till 1076mPa*6. This demonstrated that applying the shear rate up to 100 1/s decreases the viscosity of the gel (Figure 10).

Figure 7. Rheological behavior of hydrogel injection storage modulus (G′) and loss modulus (G″) of the hydrogel injection as a function of temperature.

Figure 8. Incipient gel point or sol-gel transition of hydrogel

Figure 9. The viscosity curve of the Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel
within increasing shear rate from 0.1 to 100 1/s.

SEM Imaging of hydrogels

Hydrogel pores must be tiny enough to encapsulate bacterial cells efficiently and prevent their uncontrolled migration to the outer environments [6]. The pores must also be large enough to permit macromolecule exchange between the encapsulated bacteria and the surrounding tumor microenvironment. The average pore size of Chitosan/β-glycerophosphate hydrogel equals 56.61 ± 18.82 μm [7]. It is too large for efficient trapping of E. coli cells since such big pores will enable bacterial migration from the gel. Initially, we added 4% (w/w) SH to the CH/GP hydrogel, intending to decrease the pore size. SEM images of Chitosan/β-glycerophosphate/Sodium Hyaluronate (4%) can be found in Figure 10. The average pore size was equal to 27.822 µM. This reduction is notable yet insufficient. As a result, we proceeded with further pore size optimization by adding different percentages of hyaluronic acid.

Figure 10. Rheological behavior of hydrogel injection storage modulus (G′) and loss modulus (G″)
of the hydrogel injection as a function of temperature.

As shown in Table 1, the same percentage of hyaluronic acid led to a drastic reduction of the pore size, so we did not see any of them on SEM Images of the gel (Figure 15). Therefore, cross-linking between sodium hyaluronate and β-glycerophosphate is lower than between hyaluronic acid and β-glycerophosphate. As demonstrated in Figure 15, Chitosan/β-glycerophosphate/Hyaluronic acid (4% w/w) hydrogel has a film-like appearance. We did not observe any pores, even at 5.00 K X magnification. It is possible that the addition of hyaluronic acid (4% w/w) reduced the pore size to a nanometer scale. However, it was impossible to test this hypothesis because when we aimed for greater magnification, the gel began to change its morphology and form bubbles due to electron bombardment of a small area. We can conclude that hyaluronic acid, even in relatively small quantities, can significantly reduce the pore size of the Chitosan/Glycerophosphate hydrogel. Lack of pores would substantially reduce (and possibly eliminate) bacterial proliferation since macromolecule exchange would be inhibited in such gels. No pores were observed on Chitosan/β-glycerophosphate/Hyaluronic acid (3% w/w) hydrogel (Figure 14).

Table 1. Correlation of the pore sizes of CH/GP/HA hydrogels to the proportion of hyaluronic acid.

CH proportion	GP proportion	HA proportion	Total DI Water	Average pore size
60 mg	300 mg	0 mg	2.5 mL	56.61 ± 18.82 μm [7]
60 mg	300 mg	1.875 mg (0.5% w/w)	2.5 mL	18.39 μm
60 mg	300 mg	3.75 mg (1% w/w)	2.5 mL	11.54 μm
60 mg	300 mg	7.5 mg (2% w/w)	2.5 mL	3.94 μm
60 mg	300 mg	11.25 mg (3% w/w)	2.5 mL	Not Observed
60 mg	300 mg	15 mg (4% w/w)	2.5 mL	Not Observed

The results of the SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (0.5% w/w) hydrogel are illustrated in Figure 11. The average pore size of this hydrogel is equal to 18.39 μm. Figure 12 contains the SEM image of Chitosan/β-glycerophosphate/Hyaluronic acid (1% w/w) hydrogel, which has an average pore size of 11.54 μm. Hence, these two formulations were eliminated from further experiments as the pores could allow high rates of bacterial migration. Figure 13 demonstrates SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (2% w/w) hydrogel. The average pore size of this hydrogel is equal to 3.94 μm. So far, this data was the most fitting for the sustainment of the objectives of our system. Because of this, this formulation was chosen for further studies and bacterial encapsulation. To evaluate whether this hydrogel would minimize bacterial leakage, the Bacterial Release Test was performed. Other hydrogels containing different percentages of hyaluronic acid between 2% and 3% could be synthesized in future experiments to select the most optimal formulation.

Figure 11. SEM Imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (0.5% w/w) hydrogel.

Figure 12. SEM Imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (1% w/w) hydrogel.

Figure 13. SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (2% w/w) hydrogel.

Figure 14. SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (3% w/w) hydrogel.

Figure 15. SEM imaging of Chitosan/β-glycerophosphate/Hyaluronic acid (4% w/w) hydrogel.

Bacterial release test

One of the key reasons the system includes hydrogel is that the gel’s matrix can trap bacteria inside and prevent the uncontrolled spreading of the cells in the organism. To evaluate whether Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel can efficiently encapsulate bacteria and prevent their release into the surroundings, the Bacterial Leakage Test was conducted. The primary (raw) data of the Bacterial Release Test can be found on the Lab Notebook page, while the calibrated results are summarized in Table 2. The first measurement (1 minute after sample loading) was taken as the baseline for the hydrogel-encapsulated bacteria sample, and the rest of the data from this group was normalized accordingly. The graph demonstrating the change in the OD600 nm of free E. coli cells and Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel encapsulated E. coli cells can be found in Figure 16. As illustrated, E. coli cells could rapidly grow and increase in the media as free bacteria. However, encapsulation of the E.coli cells into Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel efficiently prevented the bacteria migration out of the hydrogel. It is essential to note that bacterial release was not observed even at longer elapsed time points (24 hours, 48 hours, and 72 hours).

Table 2. Bacterial release test calibrated data.

	Sample
Elapsed Time (hh:mm:ss)	Encapsulated E.coli	Free E.coli
00:01:00	0	0.0169
00:05:00	0.0222	0.0184
00:10:00	0.0645	0.0211
00:30:00	0.0092	0.0423
01:00:00	0.0362	0.0897
03:00:00	0.0646	0.3948
04:00:00	0.0997	0.4883
06:30:00	0.1364	0.8539
14:00:00	0.1735	1.3961
24:00:00	0.1646	1.633
48:00:00	0.1655	1.8415
72:00:00	0.1124	1.8831

Figure 16. Graph demonstrating calibrated results of the bacterial release test.

Overall, the results of this experiment demonstrate that Chitosan/β-glycerophosphate/Hyaluronic acid (2%) hydrogel can efficiently encapsulate bacteria and minimize their escape to nearby environments. Therefore, integrating such systems could reduce the side effects of conventional bacterial therapies by preventing the unwanted spread of the cells across the organism.

MTT Assay

Literature sources indicate that chitosan-based hydrogels exhibit biocompatibility and biodegradability, which allows us to view their injection into the human body as safe [1, 2]. However, the data regarding the cytotoxic effects of Chitosan/β-glycerophosphate/Hyaluronic acid hydrogel on bacterial cells was not found. Hence, an MTT Assay was conducted to evaluate whether this formulation is suitable for bacterial encapsulation. This test is necessary since, in the case of high cytotoxicity of the gel to bacteria, it cannot be used in combination with bacterial therapeutics as most of the cells will not survive, and, as a result, the payload for treatment will be minimal. Table 3 represents calibrated MTT Assay results. Raw data can be found on the Lab Notebook page. Figure 17 illustrates calibrated results in the form of box plots. The main principle of MTT Assay is based on the reaction in which nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductases reduce yellow 3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide (MTT reagent) to a formazan, which forms insoluble purple granules. Hence, this method is applied to evaluate cell viability under specific conditions (such as the presence of certain chemicals). The first column (Bacteria/LB sample) was used as a positive control in this experiment. The second column contained Bacteria/Chloramphenicol (bacteriostatic antibiotic) and served as a negative control. The last two columns were filled with LB broth and empty hydrogel. 3-(4,5-dimethylthazolk-2-yl)-2,5-diphenyl tetrazolium bromide was also added to these columns. The results of the controls satisfied our expectations since the positive control demonstrated a proper reduction of the MTT reagent by living cells. In contrast, the negative controls showed that in the absence of cells, the MTT reagent remains yellow, and the degree of color change (and, consequently, OD570 nm value) is proportional to the number of viable cells in the sample.

Figure 17. Box plot representation of calibrated MTT Assay data.

Table 3. MTT Assay calibrated data.

Sample	Bac/LB	Bac/Chl	Bac/Gel
A	1.680740875	0.765178875	0.56598775
B	1.362780875	0.973510875	1.12236775
C	1.440980875	0.726283875	1.07879775
D	1.414380875	0.790633875	0.86929775
E	1.401720875	0.672601875	1.05484775
F	1.247490875	0.895230875	0.98855775
G	1.069670875	0.973860875	0.99048775
H	1.2202709	0.826714875	2.02034775
Mean	1.373449	0.828002	1.0863365