Arrhythmic Effects Evaluated on Caenorhabditis elegans: The Case of Polypyrrole Nanoparticles

Experimental studies and clinical trials of nanoparticles for treating diseases are increasing continuously. However, the reach to the market does not correlate with these efforts due to the enormous cost, several years of development, and off-target effects like cardiotoxicity. Multicellular organisms such as the Caenorhabditis elegans (C. elegans) can bridge the gap between in vitro and vertebrate testing as they can provide extensive information on systemic toxicity and specific harmful effects through facile experimentation following 3R EU directives on animal use. Since the nematodes’ pharynx shares similarities with the human heart, we assessed the general and pharyngeal effects of drugs and polypyrrole nanoparticles (Ppy NPs) using C. elegans. The evaluation of FDA-approved drugs, such as Propranolol and Racepinephrine reproduced the arrhythmic behavior reported in humans and supported the use of this small animal model. Consequently, Ppy NPs were evaluated due to their research interest in cardiac arrhythmia treatments. The NPs’ biocompatibility was confirmed by assessing survival, growth and development, reproduction, and transgenerational toxicity in C. elegans. Interestingly, the NPs increased the pharyngeal pumping rate of C. elegans in two slow-pumping mutant strains, JD21 and DA464. Moreover, the NPs increased the pumping rate over time, which sustained up to a day post-excretion. By measuring pharyngeal calcium levels, we found that the impact of Ppy NPs on the pumping rate could be mediated through calcium signaling. Thus, evaluating arrhythmic effects in C. elegans offers a simple system to test drugs and nanoparticles, as elucidated through Ppy NPs.


Synthesis of Ppy NPs
The Polypyrrole Nanoparticles (Ppy NPs) were synthesized by chemical oxidative polymerization method (Figure S1).Briefly, pyrrole (0.1 M), being the monomer of interest, was added to a solution of FeCl 3 .6H 2 O oxidant (24:1 to monomer ratio) and PVA surfactant (7.5 wt% of monomer).The mixture was sonicated at 5 °C for 4 hours to complete the polymerization reaction and washed thrice with distilled water until a clear supernatant was observed.The pellet was redispersed in distilled water and maintained at 4 °C for future use.

Characterization of Ppy NPs
The as-synthesized Ppy NPs were dried at 60 °C to obtain dry powders for physicochemical characterization.The size and morphology of the NPs were examined by electron microscopy and dynamic light scattering (DLS) techniques.For the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analysis, aqueous dispersion of Ppy NPs were prepared at 10 µg/mL concentration, and the mean diameter was 132±31 nm.The SEM and TEM images revealed that the NPs exhibits a uniform spherical morphology (Figure S2a, b).
Likewise, for the DLS analysis, we prepared 100 µg/mL dispersion in Milli-Q water and the scan was performed with 10 runs/measurement.The hydrodynamic diameter of Ppy NPs as revealed by DLS was 196±62 nm with a polydispersity index of 0.17 (Figure S2c).The PDI corroborates well with the dispersion stability of the NPs solution (Figure S1b).The NPs´ sizes were reproducible and agreeing with the previous reports using this protocol.The chemical structure of Ppy NPs can be appreciated from the FTIR spectra (Figure S2d).The broad intense peak at 3311 cm -1 is due to N-H stretching of secondary amines and the peak at 1631 cm -1 occurs from C=C stretching of conjugated alkenes.The smaller peaks at 1324 and 862 cm -1 correspond to C-N stretching from aromatic amines and C-H bending in alkane chains, respectively.Similarly, the Ppy formation was also confirmed by UV-Vis-NIR spectra, where the NPs displayed a typical broad and intense absorbance in the NIR region (700-1200 nm).
The thermal degradation behavior of Ppy NPs (≈ 1 mg) was studied by thermo-gravimetry analysis.The NPs were heated up to 800 °C, with a heating rate of 10 °C/min.The TGA spectra shows that Ppy NPs possess exceptional thermal stability, with a gradual decomposition rate and maximum degradation between 200-400 °C.

Recovery of Ingested NPs
After employing the bleaching protocol to digest worms and recover the ingested NPs, the sample is cleaned thoroughly with Milli-Q water and immediately visualized in optical microscopy (Figure S3) prior to TEM analysis, to ensure that the worms are digested, but the NPs are present.The hydrodynamic diameter and polydispersity index are also measured to study the aggregation behaviour of the ingested NPs and found to be 311±75 nm and 0.3, respectively.

Survival rate
The worms exposed to 100 µM of PL and RE for 24 hours were scored for survival after exposure, to estimate whether PL and RE are compatible in C. elegans.Both the substances showed ≈99% survival, validating the suitability to be analysed in C. elegans.

Figure
Figure S1.a) Synthesis of Ppy NPs by oxidative polymerization b) Ppy NPs uniformly

Figure S2 .
Figure S2.Size and Morphology of Ppy NPs a) SEM image, b) TEM image, c) hydrodynamic

Figure S3 .
Figure S3.The ingested Ppy NPs recovered from C. elegans a) visualized by optical

Figure S4 .
Figure S4.The survival rate of C. elegans after 24 hours treatment with PL and RE at 100 µM

Table S1 .
The mean change in pumping rate (pumps/min) of Ppy NPs treated N2, JD21, and DA464 C. elegans, the rate of increase from 0 to 24 hours, and rate of decrease from 24 to 96 hours.