Introduction:: Pancreatic Ductal Adenocarcinoma (PDAC) is a fatal disease characterized by dense stroma and increased stiffness in the tumor microenvironment (TME). PDAC is known for having a stiff and complex TME making it difficult to investigate tumor progression and treatments[1]. To better understand the progression of this disease, various hydrogel systems have been used to simulate different TME. However, how stiffness heterogeneity in the TME affects tumor cell progression or migration has not been reported. Hydrogels are 3-D crosslinked polymeric materials that have a wide range of properties such as stability and stiffness[2]. Hydrogel size can vary from over one centimeter in diameter (bulk gel) to less than one millimeter in diameter (microgel). Recently, microgel has become a more attractive strategy in creating TME as it can be annealed together to create microporous annealed particle hydrogels (MAP). These MAP gels have been shown to have advantageous mass transport properties making them a better material for cell studies[3]. This study sought to help understand PDAC tumor progression by developing a material that can be annealed together to create a microgel stiffness gradient.
Materials and Methods:: First, hydrogels with various concentrations of GelNB-CH, 4-arm PEG thiol (PEG4SH), 8-arm PEG-norbornene (PEG8NB), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) were tested in bulk gel form using a rheometer and microtestor in order to evaluate the stability and modulus of the formulation. These tests revealed the optimal formula to be 5% GelNB-CH, 1% PEG8NB, 1% PEG4SH, and 2 mM LAP. Then microgel was synthesized using the optimal formula and stiffened for 24 hours using different concentrations of oxidized dextran (0%, 0.1%, 0.5%, and 1% oDex). After 24 hours the oDex solution was removed along with a small portion of the microgel for imaging and microgel size measurements. Once removed the remainder of the microgel was annealed overnight in multiple 10 mm cylindrical 3-D printed molds using 0.5% PEG-tetrazine (PEG-Tz) through inverse electron demand Diels-Alder (iEDDA) reaction. At least 3 MAP gels were made for each concentration of oDex. Once the microgel were fully annealed, they were removed from the mold and the storage modulus was tested using a rheometer. Both microgel diameter and storage modulus data were collected every day for seven days.
Results, Conclusions, and Discussions:: For microgel size measurements, at least 50 samples were measured from each stiffness group. The unstiffened and 0.1% oDex stiffened microgels were significantly (p< 0.05) larger than the 0.5% and 1% oDex stiffened microgel. The unstiffened and 0.1% oDex stiffened microgels were not significantly (p > 0.05) different in size. Also, the 0.5% and 1% stiffened microgels were not significantly different in size. The concentration of oDex used for stiffening had a significant effect on the MAP gel storage modulus. The Map gel storage modulus is dependent on the concentration of oDex used for stiffening. Also, the storage modulus remained stable throughout the seven-day period. The results demonstrate the concentration of oDex used for microgel stiffening significantly affects the size of the microgel and the storage modulus of the MAP gel they are used to create. Higher concentrations of oDex such as 2% were tested in earlier groups, however, it did not increase the storage modulus of MAP gel or change the size of the microgel significantly when compared to the 1% oDex stiffened microgel. This study was limited in that only a limited number of MAP gels were synthesized and tested. Future studies will look to anneal the different groups of stiffened microgel together in order to create a smooth gradient for cell study.
Acknowledgements (Optional): : This study was supported by NSF REU program 1950672.
References (Optional): : 1Sarantis P et al., World J Gastrointest Oncol 2020. 2Chang C et al., Acta Biomaterialia 2021. 3Jiang Z et al., Biomaterials Advances 2022.
