Associate Professor University of Florida, United States
Introduction: Autism spectrum disorder (ASD) affects around one in every 36 children in the United States, with an estimated $461 billion annual economic burden (1, 2). Although the causes of ASD are still unknown, it is becoming clear that ASD is more than just a behavioral issue but rather a complex and extremely heterogeneous biological disorder. Until recently, the brain was believed to be "immune privileged." However, it is now understood that the immune system plays crucial roles in the development and function of the brain throughout life. Specific to the mechanism of maternal autoantibody-related (MAR) ASD, brain-reactive maternal autoantibodies cross the blood-brain barrier during gestation and bind to the various autoantigens, including lactose dehydrogenase B (LDH-B), in developing neurons. Upon binding to their intracellular targets, these maternal autoantibodies elicit adverse effects to neurodevelopment such as a significant decrease in the number and density of dendritic spines on neurons in the developing cortex which have been linked to MARA (3). Herein, we propose the development of Systems for Nanoparticle-based Autoantibody Reception and Entrapment (SNAREs), which we believe will be capable of removing disease-causing MAR autoantibodies from maternal blood prior to their accumulation in the fetal brain. Here, we synthesized SNAREs by surface modification of mPLGA NPs with PEG-amines (H2N-PEG-NH2/CH3O-PEG-NH2) and MAR peptides. In addition, we tested them in vitro for cytotoxicity and their ability to capture LDH-B antibodies in maternal blood.
Synthesis NPs: PLGA NPs were prepared using a bulk nanoprecipitation method with a small modification to a previous published method (3). Briefly, PLGA (100 mg) was dissolved in ACN (10 mL) and mixed with 4% aqueous ammonium bicarbonate (1 mL) until a clear solution was obtained. Then it was added dropwise to a beaker containing 100 mL of PVA (2% w/v in ultra-pure water), and the solvent was evaporated during magnetic stirring for 16 h. The resulting PLGA nanoparticles were washed twice with ultra-pure water by centrifugation at 18000 RPM for 10 min at room temperature, and the lyophilized NPs were stored at -20 °C. Surface modification: PLGA NPs (25 mg) were resuspended in PBS (1 mL, 1 M in 0.2% tween20) and incubated at 37 °C with EDC (25 mg) and NHS (30 mg) for 12 h. Then, PEG-diamine (100 mg) was added to the activated NP solution, and the resulting suspension stirred for 48 h on a magnetic stirrer at room temperature. And the PEGylated NPs were separated by centrifugation at 10 kRPM for 5 min and washed 3 times with DI water, followed by overnight lyophilization.
Results, Conclusions, and Discussions: Results and
Discussion: After the synthesis, mPLGA NPs were analyzed by dynamic light scattering (DLS), Zeta potential reading and Scanning electron microscope (SEM). The DLS analysis showed a hydrodynamic diameter of 210 nm with a PDI of 0.015 (Figure 1, A), and it also revealed that the zeta potential corresponding to COOH groups present on NPs showed -19.5 mV (Figure 1, B). Further, the SEM analysis confirmed that the NPs have a spherical shape with a small porous surface and an average porous size of 5 nm (Figure 1, C and D). Next, we used these NPs for surface modification with both PEG-amines H2N-PEG-NH2 & MeO-PEG-NH2 in the presence of EDC and NHS, and the PEGylated NPs were confirmed by DLS-observed changes in Zeta potential from -19.5 to -0.28 (Figure 1, E). The above results indicate that the synthesized mPLGA NPs showed prominent size, PDI, zetapotential, and mesoporous properties on the surface.
Conclusion and Future Directions: The above results suggest that we synthesized both mPLGA NPs and mPLGA-NHS NPs which have desirable hydrodynamic diameters, PDI, zeta potential, and porosity. In future studies, we will further modify the surface of the mPLGA-NHS NPs with PEG-diamines before conjugating with MAR-peptides. Finally, the MAR-peptide conjugated PLGA NPs (SNARES) will be tested in vitro and in vivo for their ability to sequester pathogenic antibodies.
References: 1.Matthew J Maenner, et al. MWR Surveill Summ,. 2023, 24, 72, 1-14. 2. Mark Blaxill et all, J Autism Dev Disord. 2022, 52, 6, 2627–2643. 3. Karen L. Jones et all, Molecular Psychiatry, 2019, 24, 252–265. 4. Sarah Strecka et all, International Journal of Pharmaceutics: X 1,2019, 100030.
