Troubleshooting and protocols optimization
As we have worked with the hydrogel, assessment of its parameters was crucial. To test hydrogel properties and interactions with bacteria, we have designed and optimized subsequent protocols:
Bacterial release protocol
Previously, other teams worked on developing methods for measuring the bacterial escape rate from shell-coated hydrogels. However, such protocols can not be applied to different hydrogels, such as in situ hydrogels that do not contain additional coating. To address this challenge, we designed and optimized the protocol to evaluate the bacterial release rate from such hydrogels. This protocol can be helpful when hydrogel does not contain a rigid capsule. Detailed protocol of this contribution can be found in the Experiments section.
MTT Assay
MTT Assay was initially designed and is commonly used to evaluate the cytotoxicity of chemicals on eukaryotic cells. Since we aimed to determine the toxicity degree of the synthesized hydrogel on bacterial cells and consider whether this formulation can be used for bacterial therapeutics, we modified and optimized the MTT proliferation assay protocol to suit bacterial cells. Detailed protocol of this contribution can be found in the Experiments section.
Chitosan-based hydrogel characterization and optimization for bacterial encapsulation
Throughout the summer, we worked on evaluating the effect of Sodium Hyaluronate and Hyaluronic Acid on the pore size of Chitosan/Beta-Glycerophosphate-based hydrogels. One of the essential characteristics of hydrogels is their pore sizes. This parameter must be precisely tuned to fit the objectives of the system. In our case, we tested various chemical compositions to decrease the pore size of our hydrogel to efficiently prevent bacterial escape and allow for a high rate of macromolecule exchange between the encapsulated bacteria and tumor microenvironment. We performed several trials of hydrogel synthesis with adjusted formulations. Different concentrations of Sodium Hyaluronate and Hyaluronic Acid were added to the gel, and the pore size change was detected via SEM microscopy. As a result, we could determine and describe the effect of these cross-linking agents on the pore size of Chitosan/Beta-Glycerophosphate-based hydrogels.
HYDROGEL GUIDEBOOK
Additionally, we evaluated the difference in the properties of chitosan/glycerol and chitosan/Beta-Glycerophosphate hydrogels. We prepared a detailed guidebook for the hydrogel synthesis optimization for future teams. This data will help future teams working with hydrogels to adjust their properties to their project aims.
Safety
Working with bacteria and cancer cell lines poses some safety issues that are very crucial to address. Our safety page describes all the measures to prevent contamination and protect laboratory personnel from hazards. Detailed information is on the Safety page.
