(L-445) Development of a novel emodin drug depot

Introduction:: Emodin is a natural plant derivative with many therapeutic properties including anti-cancer, anti-apoptosis, and anti-inflammatory effects. However, the delivery of emodin is quite challenging due to its super hydrophobic properties. Furthermore, conventional systemic delivery approaches often result in side effects. Thus, alternative strategies are important for successful drug delivery. The goal of this study was to develop a novel emodin drug depot for cancer drug delivery utilizing peptide amyloids. For the peptides, an aggregation-prone amino acid domain of receptor-interacting serine/threonine-protein kinase 3 (RIP3) protein was used. The societal impact of these studies could be significant as RIP3 protein is known to play a role in necroptosis, and the overexpression of RIP3 has been shown to reduce cancer progression. Moreover, emodin has been shown to induce RIP3 expression. Hence RIP3 based depots to deliver emodin could provide an additional advantage in serving as drug depots. The RIP3/emodin aggregates physicochemical characterization, cellular uptake, and effects on toxicity/necrosis, are being currently studied. Preliminary studies reveal that emodin-encapsulated RIP3 peptide amyloid aggregates were able to induce significant cancer cells toxicity compared to free emodin. Hence, RIP3 peptide aggregates could have the potential as delivery vesicles.

Materials and Methods:: RIP3 peptide (NIYNCSGVQVGD) was custom synthesized from Genscript, New Jersey. All other chemicals were purchased from Sigma Aldrich and/or Fisher Scientific. Aggregates were produced by dissolving 20μl Emodin (1 mg/mL) in a 200μl solution produced by adding 2μl of RIP3 in 1mL of pH7 100 mM Ammonium Acetate buffer. This created a desired concentration of 100 μM that was then incubated at 37°C overnight. The data is represented as mean standard error (S.E.). The aggregates were then concentrated using a 3k MWCO before physicochemical characterization of the amyloid aggregates, and cellular efficacy studies were performed. Aggregation was confirmed by thioflavin T (ThT) fluorescence data at 440/482 nm. The particle size was measured by dynamic light scattering (DLS), and electron microscopy images. Emodin encapsulation was determined by absorbance measurements at 450 nm using a spectramax spectrophotometer, on a developed Emodin calibration curve. Cancer cells toxicity effects were performed by alamarBlue assay measured at an excitation wavelength of 570/590 nm. This data was used to determine the extent to which the Emodin served as a drug depot. Three or more independent experiments were performed to determine statistical significance, the t-test was also used to determine statistical significance and is represented as * p < 0.05, or ** p < 0.01, or *** p < 0.001.

Results, Conclusions, and Discussions:: Results

Peptide aggregates showed potential as drug depots for hydrophobic drug delivery as demonstrated. DLS demonstrated shifts in size from our control particles to our Emodin delivery particles as noted in Figure 1A. This data exhibits the encapsulation of the Emodin particles. The aggregates exhibited size in the range of 600-1100 nm. Peptide aggregation was confirmed by ThT data noted in Figure 1B. The emodin encapsulation efficiency was determined to be around 25% utilizing Figure 1C. Particles alone did not show significant toxicity, as noted in Figure 1D through the comparison of the 5μg/mL Emodin to the 5μg/mL RIP3/Emodin sample as well as the 10μg/mL Emodin to the 10μg/mL RIP3/Emodin sample. The comparisons of the data yielded positive results as both RIP3 samples within the A549 lung cancer cells in which they were tested, showed increased cellular toxicity compared to emodin alone.



Conclusions

Current results show that encapsulating emodin with peptide amyloids yields a much more profound and positive effect on cancer cells than emodin alone. Results show that not only are the peptide amyloids serving as a depot for the emodin particles, as well as allowing the emodin to have a higher efficacy. These results demonstrate the need and importance of developing drug depots. We anticipate further optimization of our processes and improving the emodin efficacy of the formulation by fine-tuning the properties of the aggregates. Current data has yielded headway into this process through demonstrated efficacy and toxicity results. In addition, we plan to investigate whether the aggregates play a role in the necroptosis process, RIP3 expression, and immune response.



Discussions

Though still in the early stages, our study shows that RIP3 peptide-based amyloid aggregates have potential as drug depots. In addition, peptide-based aggregates have the added advantage of polymeric nanoformulations due to their intrinsic therapeutic properties. RIP3 protein is essential for necroptosis and hence a beneficial amyloid. In addition to enhancing cellular uptake, a cell-penetrating peptide sequence could be added to the peptide domain. Optimization on particle size, site-specificity, are some of the parameters that could be additionally investigated for a successful depot.


Acknowledgements (Optional): : We would like to thank NIH R15GM135766, and the University of Michigan-Dearborn for funding.

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