Single-Neuron Gene Delivery via a PDMS-Based ...

Single-Neuron Gene Delivery via a PDMS-Based

Multielectrode Array

Device Overview

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Integrated glass micropipette for gene delivery

Implantable PDMS and gold-based microelectrode array for electroporation

Contained system targeting single neurons in vivo

Purpose

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Neuromodulation

"the alteration of nerve activity through targeted delivery of a

stimulus, such as electrical stimulation or chemical agents, to specific neurological sites in

the body"

Target

Interest to modulate certain neurons’

phenotype

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Goal

Eventual use in research and to treat disease

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How Will This Be Accomplished?

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ElectroporationDifferent methods and

general application

Gene Transfection

Viral and non-viral methods of gene delivery

Microelectrode ArrayTypes of popular models used

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Electroporation: Definition

● Gene delivery through the induction of an electric field to disrupt the cell membrane and allow the movement of genes/drugs into the cell

● Operate at a specific values ○ Adjust pressure/speed of electrode penetration to reach target depth of neuron

● Include ECM-disrupting enzymes to improve transduction efficiency

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Electroporation: Methods

● Single-cell electroporation for gene transfer○ Direct microstimulation to deliver genes and other molecules with a micropipette to alter

phenotypes of single neuron cells○ Glass micropipette tip with diameter of less than 1 micron

● Combination of electroporation and single-neuron recording○ Uses recorded action potentials and electrode resistance○ Optimizes the degree of contact between the electrode tip and target neuron to increase efficiency

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Microelectrode Arrays

● Monitor and record electrophysiological signals ○ Transduce change in voltage from the flow of ions to read signals○ Ability to monitor excitation and depolarization of target cells

● Michigan Model ○ Silicon-based with variable design options○ Higher density of sensors ○ Obtain data along the length of the probe

■ Improves spatial resolution

● Utah Model○ Measure signal at the tip of probe

● Flexible arrays ○ Reduces damage to surrounding tissue

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Genetic Transfection

● Targeted gene delivery: introduce desired genetic material to target while sparing neighboring cells

● Genetic material delivered to the cell nucleus○ Viral vector gene carriers

■ Retroviral, lentiviral, and Adeno-associated○ Bacterial gene carriers

■ Plasmid DNA○ Other methods

■ Calcium phosphate, lipid, or protein complexes, polymer-mediated, electroporation or microinjection.

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Proposed Device

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Structure

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Fabrication of Plateau MEA

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Fabrication of MEA with micropipette

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Incorporation of Gene Delivery System

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Biocompatibility

● Modify device with polydopamine (PDA) and hyaluronic acid (HA) to increase biocompatibility○ PDA acts as a mediator○ HA suppresses inflammation and

coagulation

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Safety Testing

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Test for inflammatory response

● In vitro with neutrophils and macrophages

● In vivo with animal

Biocompatibility TestingISO 10993

● Genotoxicity● Carcinogenicity● Cytotoxicity

Hemocompatibility Testing

● Confirm that there is no clotting

Functional Testing

● Determine the resistance of the electrode tip○ Too high = electrode could damage cell membrane○ Too low = electroporation cannot occur

● Determine efficiency of transfection○ In vitro

■ Gene delivered should contain fluorescent marker

■ Flow cytometry to detect marker○ in vivo

■ Employ gene delivery mechanism in animal■ Examine tissue for transfection

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Possible Applications

1 Fluorescent labeling to track changes in morphology

2 Examine function of certain genes in vivo for insights into nervous system function

3 Study relationships between neurochemistry, morphology, and cell behavior

4 Control protein expression as a treatment of disease→ Parkinson’s→ Alzheimer’s→ Creutzfeldt-Jakob

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Limitations

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Efficiency of transfection

MinimallyInvasiveDelivery

Implantation Accuracy

Future Directions

1 Stand-alone integrated system→ Time studies on reservoir volume

2 Tether into soft brain tissue

3 Multi-branched system → Single reservoir→ Branching catheter or arrays

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References1. Breakefield, X. O., & DeLuca, N. A. (1991). Herpes simplex virus for gene delivery to neurons. The new biologist, 3(3), 203-218.2. Mali, S. (2013). Delivery systems for gene therapy. Indian journal of human genetics, 19(1), 3.3. Dempsey, B., Turner, A. J., Le, S., Sun, Q. J., Bou Farah, L., Allen, A. M., ... & McMullan, S. (2015). Recording, labeling, and transfection of single neurons in deep brain structures. Physiological

reports, 3(1).4. Haas, K., Sin, W. C., Javaherian, A., Li, Z., & Cline, H. T. (2001). Single-cell electroporation for gene transfer in vivo. Neuron, 29(3), 583-591.5. Oyama, K., Ohara, S., Sato, S., Karube, F., Fujiyama, F., Isomura, Y., ... & Tsutsui, K. I. (2013). Long-lasting single-neuron labeling by in vivo electroporation without microscopic guidance.

Journal of neuroscience methods, 218(2), 139-147.6. Wanisch, K., Kovac, S., & Schorge, S. (2013). Tackling obstacles for gene therapy targeting neurons: disrupting perineural nets with hyaluronidase improves transduction. PloS one, 8(1), e53269.7. Osten, P., Dittgen, T., & Licznerski, P. (2006). 13 Lentivirus-Based Genetic Manipulations in Neurons In Vivo. The dynamic synapse: molecular methods in ionotropic receptor biology, 249.8. Boven, K. H., Fejtl, M., Möller, A., Nisch, W., & Stett, A. (2006). On Micro-Electrode Array Revival. Advances in Network Electrophysiology Using Multi-Electrode Arrays, 24-37. 9. Cheung, K. C. (2007). Implantable microscale neural interfaces. Biomedical Microdevices 9(6), 923-938.10. Blau, A., Murr, A., Wolff, S., Sernagor, E., Medini, P., lurilli, G., … & Benfenati, F. (2011). Flexible, all-polymer microelectrode arrays for the capture of cardiac and neuronal signals. Biomaterials,

32(7), 1778-1786. 11. Kim, J.M., Im, C., & Lee, W. R. (2017). Plateau-Shaped Flexible Polymer Microelectrode Array for Neural Recording. Polymers, 9(12), 690. 12. Loeb, G., Peck, R., & Martyniuk, J. (1995). Toward the ultimate metal microelectrode. Journal of Neuroscience Methods, 63, 175-183. 13. Wise, K., Sodagar, A., Yao, Y., Gulari, M., Perlin, G., & Najafi, K. (2008). Microelectrodes, microelectronics, and implantable neural microsystems. Proceedings of the IEEE, 96, 1184-1202. 14. Metallo, C., White, R., & Trimmer, B. (2011). Flexible parylene based microelectrode arrays for high resolution EMG recordings in freely moving small animals. Journal of Neuroscience Methods,

195, 176-184. 15. Normann, R. (2007). Technology Insight: Future Neuroprosthetic Therapies for Disorders of the Nervous System. Nature Clinical Practice Neurology, 3, 444-452. 16. Wise, K. D., & Angell, J. B. (1975). A low-capacitance multielectrode probe for use in extracellular neurophysiology. IEEE Transactions on Biomedical Engineering, (3), 212-219. 17. Xue, P., Li, Q., Li, Y., Sun, L., Zhang, L., Xu, Z., & Kang, Y. (2017). Surface Modification of Poly (dimethylsiloxane) with Polydopamine and Hyaluronic Acid To Enhance Hemocompatibility for

Potential Applications in Medical Implants or Devices. ACS applied materials & interfaces, 9(39), 33632-33644.18. Wildgruber, M., Lueg, C., Borgmeyer, S., Karimov, I., Braun, U., Kiechle, M., ... & Berger, H. (2016). Polyurethane versus silicone catheters for central venous port devices implanted at the

forearm. European Journal of Cancer, 59, 113-124.

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Questions?

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