Undergraduate Purdue University Vincennes, Indiana, United States
Introduction:: Subcutaneous (SC) tissue injections are growing in popularity as a drug delivery method. SC injections can be self-administered at home, minimize infection risk, and minimize healthcare personnel resources. Insulin, growth hormones, pain medication, and vaccines utilize SC injections. These injections can be administered in the upper arm, abdomen, thigh, or upper buttocks. In the past, it was assumed that SC tissue is homogenous and that the location of the injection would play no role in drug absorption. However, it has recently been discovered that drug absorption and action vary based upon location. For example, insulin absorption is much better in the thigh when compared to the abdomen. It is unclear why regional differences affect absorption. Prior work in our lab has lead us to believe that compositional differences may be a driving factor behind drug uptake. Preliminary work showed that SC tissue is heterogenous across the belly, breast, and neck in a pig model. Currently, in order to observe this relationship in humans, the GenoSkin SC injection model from HypoSkin can be used. However, this model features SC tissue predominantly from white females with above average BMIs and the samples are very expensive. Overall, there needs to be a better form of SC tissue collection in humans in order to study drug absorption variability. We hypothesize that a 14-gauge core needle biopsy can be used to collect sufficient tissue mass for LC-MS/MS protein characterization.
Materials and Methods:: Using tissue samples collected from the belly of a Yucatan mini pig, three SC tissue samples were collected using the 14-gauge core needle biopsy (masses around 10mg) and three 100 mg SC tissue samples were dissected. The whole tissue samples were then homogenized. The tissue homogenates underwent chloroform-methanol precipitation in order to remove the lipids. The resuspended tissue homogenate was ran through a LDS-PAGE to filter and in-gel digestion methods were utilized. First, the gel bands were excised and then digested using trypsin. The resulting peptides were then desalted using spin columns to remove harmful salts for LC-MS/MS. Prior to LC-MS/MS, the protein in the samples were quantified using a micro-BCA.
Results, Conclusions, and Discussions:: The LDS-PAGE and micro BCA showed an abundance of proteins in each sample upstream of mass spectrometry. Using the peptide LFQ intensities, the Pearson r correlation coefficients were calculated. There were strong protein composition differences within sample groups and across sample groups. These correlation differences within groups are likely due to tissue heterogeneity. A volcano plot is used to show the statistically and biologically different proteins between sample groups. There were many proteins identified to be upregulated in the 100mg dissected samples. Overall, the core needle biopsy successfully collected enough SC tissue for LC-MS/MS analysis. However, tissue heterogeneity was shown in the samples, likely due to the striation of belly tissue. In the future, this study will be repeated using samples from pig neck in order to reduce the tissue heterogeneity. In-solution digestion methods will also be utilized instead of in-gel digestion as the in-gel digestion may have eliminated some proteins.
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References (Optional): : 1] Roman Mathaes et al., “Subcutaneous injection volume of biopharmaceuticals-pushing the boundaries,” Journal of Pharmaceutical Sciences, https://www.sciencedirect.com/science/article/pii/S0022354916414656?casa_token=PNxRSqY7XbIAAAAA%3A-Kko16S83QYOu1KGdTGC6GJo3SqRz0EACJPkrKMp5njkO36lrkilAIm5HuWoMTa6znlrgW7D-dA (accessed Jul. 17, 2023).
[2] H. Kim, H. Park, and S. J. Lee, “Effective method for drug injection into subcutaneous tissue,” Scientific reports, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575294/#:~:text=As%20subcutaneous%20tissue%20has%20few,at%20a%20low%20dose%20rate. (accessed Jul. 17, 2023).
