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Experimental Design

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Epilepsy and Parkinson's disease are two prevalent neurological diseases currently plaguing many people that are caused due to abnormal or decreased neural activity (2,4). Among the currently available treatments, electrical stimulation and optogenetics has the highest efficacy rate and most effective treatment approaches but requires surgical procedures to implant devices in the brain that are damaging to biological tissues. Recently, magnetothermal therapy has been widely explored for its application in neural activity modulation. The compound capsaicin, a neuron modulary drug found in chili peppers, has been studied for its effects and benefits and is known to activate sensory neurons (1).

The research team led by Dr. Romero has developed a magnetothermal soft material system that delivers dopamine to stimulate striatal neurons. It consists of a core made of Iron oxide nanoparticle and a coating of POEGMA brushes, a biocompatible, thermo-responsive polymer whose response is triggered by change in local temperature (3).

The current study is designed to test the capability of the developed drug delivery system in delivering capsaicin to stimulate hippocampal neurons. Neurons will be exposed to four different experimental conditions including (Alternating Magnetic Field) AMF and/or capsaicin loaded nanoparticles. Neurons in the fourth group are exposed to both AMF and capsaicin loaded nanoparticles. Applied AMF triggers the thermodynamic response of POEGMA brushes grafted over nanoparticles, thus releasing the encapsulated capsaicin.

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Introduction

Hippocampal Neuron Stimulation By Local Capsaicin Release

Sean Macias, Rohini Thevi Guntnur, PhD, Gabriela Romero, PhD

Department of Biomedical Engineering, University of Texas at San Antonio

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Conclusion

Results

• A wider size distribution observed in DLS measurements for nanoparticles with POEGMA brushes, corresponds to the grafting of the POEGMA brushes from the surface of Iron Oxide Nanoparticles.
• Surface Functionalization is observed to influence the surface charge on nanoparticles as seen in ζ-potential measurements.
• The heating efficiency of the particles is seen to be affected by the surface functionalization with POEGMA brushes.
• The Neuron ID graphs show the controlled stimulation of hippocampal neurons due to local release of capsaicin on exposure to AMF. From the summary bar graph, it is observed that the experimental group was able to stimulate approximately 70% of the neurons which is equivalent to the activity observed in the positive control group with capsaicin alone.
• The lower neural activity seen in the control group confirms that the activity seen in the experimental groups is due to specific interaction between the locally released capsaicin molecules and TRPV membrane receptors on the hippocampal neurons.
• Further, the excitation of the hippocampal neurons due to magnetothermal release of capsaicin can be observed by the increase in the fluorescence intensity as represented by the fluorescence images taken from calcium imaging videos.
• Overall, this indicates that the iron oxide nanoparticles system is effective in the local delivery of capsaicin to stimulate hippocampal neurons.

References

1. Fattori, V., Hohmann M.S.N., Rossaneis A.C., Pinho-Ribeiro F.A., & Verri, W.A. (2016). Capsaicin: Current Understanding of Its Mechanisms and Therapy of Pain and Other Pre-Clinical and Clinical Uses. Molecules, 21(7), 844. https://doi.org/10.3390/molecules21070844
2. Guekht, A., Brodie, M., Secco, M., Li, S., Volkers, N., & Wiebe, S. (2021). The road to a World Health Organization global action plan on epilepsy and other neurological disorders. Epilepsia, 62(5), 1057–1063. https://doi.org/10.1111/epi.16856
3. Guntnur, R.T., Muzzio, N., Morales, M., & Romero, G. (2021). Phase transition characterization of poly(oligo(ethylene glycol)methyl ether methacrylate) brushes using the quartz crystal microbalance with dissipation. Soft Matter, 9, 2530-2538. https://doi.org/10.1039/D0SM02169E
4. Reeve, A., Simcox, E., & Turnbull, D. (2014). Ageing and Parkinson's disease: why is advancing age the biggest risk factor?. Ageing research reviews, 14(100), 19–30. https://doi.org/10.1016/j.arr.2014.01.004
5. Romero, G., Christiansen, M.G., Barbosa L.G., Francisco, G., & Anikeeva, P. (2016). Localized Excitation of Neural Activity via Rapid Magnetothermal Drug Release. Advanced Functional Materials, 26 (35), 6471-6478. https://doi.org/10.1002/adfm.201602189

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Table 1: The five different test groups with each of their variables: Alternating Magnetic Frequency (AMF), Magnetic Nanoparticles (MNP), and Capsaicin.

Acknowledgements

I would like to thank Dr. Guntnur for helping me throughout my project with the methods and interpretations of my results and Dr. Romero for welcoming me into her lab and allowing me this opportunity.

Figure 3: Graphs representing the neural activity observed in different groups: A) Negative Control B) Positive Control-Capsaicin Only C) Experimental Test Group. The shaded regions represents the application of the AMF. Dots on the graph indicate the neural activity of 100 random neurons picked by the MATLAB code showing the activity stimulated at respective times.

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While the POEGMA brushes were grown over nanoparticles using ATrP technique, the hydrophobic and steric interactions between the capsaicin molecules and the polymer strands were used to encapsulate capsaicin into POEGMA brushes. The nanoparticle system was characterized for physical, morphological changes after surface functionalization via the DLS and zeta potential measurements. Further, nanoparticles were also characterized for their magnetic properties using calorimetric measurements using Magnetic Hyperthermia Machine.

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Figure 1: Schematic representation of the collapse of thermo responsive polymer brushes due to AMF, releasing the capsaicin molecules. (5)

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When the Capsaicin loaded nanoparticles are exposed to Alternating Magnetic Field (AMF), the iron oxide nanoparticle undergoes hysteresis power loss leading to the increase in the local temperature. This change in the local temperature triggers the conformational changes of the thermo responsive POEGMA brushes leading to the release of drugs locally. The specific interaction between the capsaicin and the TRPV membrane receptors will trigger the neural stimulation. Thus, the neuromodulation is achieved via controlled local release of capsaicin.

Morphological and Magnetic Characterization of Nanoparticles

Figure 2: Morphological and Magnetic characterization of Nanoparticles before and after surface functionalization: A) The hydrodynamic size distribution of nanoparticles, B) ζ-Potential measurements of nanoparticles, C) Magnetic heating efficiency of nanoparticles via Specific Loss Power (SLP) measurements.

The local release of capsaicin from particles on exposure to AMF was tested on hippocampal neurons using the calcium imaging. Fluorescence videos were recorded during the experiment were utilized to identify and quantify the neural activity observed in different conditions. Analysis was performed using MATLAB code developed by the research team.

Hypothesis

Neural Stimulation represented by Neuron ID Graphs

Excitation of Hippocampal Neurons by Capsaicin Release

Figure 4: Fluorescence images of Hippocampal Neurons treated with capsaicin loaded nanoparticles: A) Before Capsaicin release (AMF OFF), B) After Capsaicin release (AMF ON), C) Comparison of percent of responsive neurons in each group.

Experimental Test Groups

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Magnetothermal Release of Capsaicin