Genetically modifying cells has been a successful method of inducing new biochemical functions into living cells. But creating structural changes in cells can be quite challenging.

By combining genetic engineering with polymer chemistry, scientists from Stanford University wanted to design biocompatible in vivo synthesis of electroactive polymers within genetically specified cells of living animals. They leveraged existing complex cellular architectures to synthesize, fabricate, and assemble bioelectric materials that could alter the function of cells.

In the current study, researchers began by culturing rat hippocampal neurons transduced with adeno-associated virus (AAV) vectors containing humanized ascorbate peroxidase (Apex2) fused with enhanced yellow fluorescent protein for tracking.

The Stanford researchers designed a single-enzyme-facilitated polymerization system that uses chemically modified monomers of conductive polyaniline (PANI) and nonconductive poly(3,3-diaminobenzidine) (PDAB) for which polymerization is triggered by Apex2 in neurons.

This process was achieved in two steps. First, small molecule conductive-polymer precursors (N-phenylenediamine, an aniline dimer) capable of diffusion of intact tissues were perfused into experimental neurons containing Apex2. Next, oxidative radical cation polymerization was induced by adding an extremely low, nonlethal dose of hydrogen peroxide (0.1 nM H₂O₂). Apex2, a peroxidase, in the presence of radical cations and the polymers precursors synthesized conductive polymers that are deposited on targeted cells.

The polymers modified the properties of neurons depending on how they were formed -- with conductive (PANI) or nonconductive (PDAB) materials. Apex2(+)/PANI neurons showed increased capacitance (or the ability to store an electric charge) and decreased action potential, whereas Apex2(+)/PDAB neurons showed decreased capacitance associated with insulating polymers.

These results were confirmed using a number of analytical and bioimaging techniques, including ultraviolet-visible-near-infrared (UV-vis-NIR) absorption spectroscopy, variable-pressure scanning electron microscopy (SEM), and transmission electron microscopy (TEM).

The golden color illustrates the deposition of biocompatible polymers on two genetically targeted neurons at right, sparing neighboring cells. Blue diamond particles represent the monomers to make the polymer diffusing globally through the tissue. The technology only enables polymers to form in targeted cells. Image courtesy of Ella Maru Studio and Yoon Seok Kim/Jia Liu of Deisseroth/Bao laboratories at Stanford University.

While the current experiments focused mainly on brain cells or neurons, GTCA should also work with other cell types.

"We've developed a technology platform that can tap into the biochemical processes of cells throughout the body," said Zhenan Bao, PhD, co-lead researcher of the study, professor and chair of chemical engineering at Stanford University, in a statement.

When the polymerization system was applied in Apex2-green fluorescent protein (GFP) on the membrane of worm (Caenorhabditis elegans) pharyngeal muscle cells, the results were consistent with the observed effects in neurons, but the experiment confirmed the long-term biocompatibility of these reactions in worms. Four weeks after Apex2 virus injection, the researchers observed high levels of Apex2-driven polymerization, with increased capacitance after PANI reaction and decreased capacitance after PDAB. Apex(2+)/PANI worms exhibited reduced pumping frequency of pharyngeal muscle.

These results suggest a specific gain of function in living animals and an opposite-direction effect compared with conducting polymer assembly as seen by impacts on electrophysiology.

In the future, the researchers would like to explore variants of their cell-targeted technology. GTCA could be used to produce a wide range of functional materials, implemented by diverse chemical signals.

"We're imagining a whole world of possibilities at this new interface of chemistry and biology," said Dr. Karl Deisseroth, PhD, co-lead researcher and a professor of bioengineering and of psychiatry and behavioral sciences at Stanford.

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