Associate Professor University of Michigan-Dearborn, United States
Introduction: Lysozyme is the cornerstone of the innate immune system. Lysozyme has anti-microbial and immunomodulator properties due to its ability to hydrolyze peptidoglycan (PG) destabilizing the cell wall of Gram-positive bacteria and causing them to lysis. Lysozyme-mediated degradation releases large amounts of pathogen-associated molecular patterns (PAMPs) which induce activation of pattern recognition receptors such as toll-like receptors (TLR) causing downstream production of strong pro-inflammatory cytokines. A PAMP released from lysozyme degradation of PG is polyinosinic: polycytidylic acid (poly(I: C) a viral double-stranded RNA (dsRNA) these PAMPs are recognized by TLR9. The pro-inflammatory properties of these molecules have been exploited in various therapeutic contexts, including their use as adjuvants to enhance immunogenicity. Recent advancements in nanotechnology offer a promising avenue for incorporating these immunostimulatory molecules. This study aims to create a tunable lysozyme protein nanoaggregate drug depot for poly(I: C). This nanoaggregate drug delivery system offers improved precision and efficacy of these molecules compared to them alone.
Materials and
Methods: Lysozyme protein was derived from chicken egg white obtained from Sigma Aldrich. All other chemicals were purchased from Sigma Aldrich, Fisher Scientific, and or Enzo Life Sciences. Aggregates were produced by dissolving 2mg of Lysozyme in a 400μl solution of pH 2 150 mM NaCl buffer this created a desired concentration of 5 mg/ml. The aggregates were incubated at 57°C for 24 hours. After this, they were concentrated and quenched with 400μl pH 7.4 0.01M PBS. Aggregation was confirmed by thioflavin T (ThT) fluorescence data at 440/482nm. Particle size was measured by transmission electron microscopy (TEM) images. Poly (I: C) encapsulation was determined using high molecular weight poly(I: C) labeled with rhodamine at an excitation-emission of 546/576nm using a spectramax spectrophotometer, on a developed Poly (I: C)-Rhodamine Calibration Curve. The cell line used to determine the extent of the nanoaggregate effects was Raw 264.7. Cellular toxicity effects were performed by alamarBlue assay measured at an excitation wavelength 570/590nm. Cellular Nitric Oxide (NO) production was performed by a Griess Reagent NO assay measuring absorbance at 570nm. Three or more independent experiments were performed to determine statistical significance, the t-test and one-way ANOVA were also used to determine statistical significance and is represented as * p < 0.05, or ** p < 0.01, or *** p < 0.001, or **** p < 0.0001.
Results, Conclusions, and Discussions: Lysozyme protein aggregates showed potential as drug depots for immunomodulation as demonstrated in the data. Lysozyme aggregation was confirmed by ThT data noted in Figure 1A. Transmission electron microscopy (TEM) images exhibited the size of the aggregates in a range of 100 to 200nm Figure 1B. Lysozyme-Poly(I: C) aggregation was confirmed by ThT data noted in Figure 2. Poly(I: C) encapsulation efficiency was determined to be around 30% utilizing Figure 3. Lysozyme alone and the drug formulations did not show significant toxicity noted in Figure 4 through the comparison of 40μg/mL Poly (I: C) to the 40μg/mL Lyz/Poly(I: C) as well as 80μg/mL Poly(I: C) to the 80 μg/mL Lyz/Poly(I: C). NO production was significantly increased noted in Figure 5 through the comparison of 40μg/mL Poly (I: C) to the 40μg/mL Lyz/Poly(I: C) as well as 80μg/mL Poly(I: C) to the 80 μg/mL Lyz/Poly(I: C). The comparison of the data yielded positive results as both Lyz/Poly(I: C) samples within Raw 264.7 cells showed increased NO production compared to lysozyme and poly(I: C) alone.
Discussions: Though still in the early stages our study shows that lysozyme-based protein nanoaggregates have potential as drug depots. Lysozyme is essential for the innate immune response and hence a beneficial aggregate for immunomodulation. In addition to enhancing immune response, it could be beneficial to test how other PAMPs bind with the aggregate and interact with other immune receptors. Optimization of site specificity and immune response are some parameters that could be additionally investigated.
Conclusions: Current results show that encapsulating poly(I: C) with Lysozyme aggregates yields much more profound and positive effects for increasing immunogenicity compared to the poly(I: C) and lysozyme aggregates alone. Current data has promising results as a novel drug delivery system for immunomodulation demonstrated in toxicity and NO production of immune cells. In addition, currently, we are investigating further how the protein aggregate delivery affects other inflammatory responses like Reactive Oxygen Species (ROS) production and cytokine releases. Further, poly(I: C) release kinetics and characterization of cellular uptake of aggregates will be done in the future.
Acknowledgements (Optional): Acknowledgements: We would like to thank NIH R15GM135766, and the University of Michigan-Dearborn for funding.
