Research ArticlePOLYMERS

Semiconducting polymers are light nanotransducers in eyeless animals

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Science Advances  25 Jan 2017:
Vol. 3, no. 1, e1601699
DOI: 10.1126/sciadv.1601699
  • Fig. 1 Characterization of P3HT-NPs and in vivo toxicological analysis.

    (A and B) Characterization of P3HT-NPs by scanning electron microscopy and dynamic light scattering (DLS) analysis. (C) Optical absorption (left axis) and photoluminescence spectra (right axis) of P3HT-NPs in an aqueous dispersion. Fluorescence emission excitation wavelength, 470 nm. (D and E) Determination of P3HT-NP toxicity endpoints in H. vulgaris. Numerical scores ranging from 10 (healthy polyp) to 0 (dead polyp) were assigned to progressive morphological changes possibly induced by P3HT-NP treatments, at the indicated doses, and recorded every 24 hours. No significant difference is evidenced between treated and untreated polyps (D). P3HT-NP treatment does not affect the reproduction rate of Hydra. The logarithmic growth curve of a treated Hydra population (red) is fully comparable to the one obtained from a control population (E) (n0 is the number of polyps at time 0, and n is the number of animals recorded at time t). (F) In vivo bright-field (left) and fluorescence (right) imaging of a living polyp treated with P3HT-NPs. Soaking the polyps with 0.25 μM P3HT-NPs causes a fluorescent staining of all tissues. NPs after a few hours appear as fluorescent spots located inside the ectodermal cells. The top images show a head with a crown of tentacles around the mouth. Bottom images show details of the tentacle tip. Scale bars, 200 and 50 μm (top and bottom images, respectively). a.u., arbitrary units.

  • Fig. 2 P3HT-NPs induce a behavioral response in H. vulgaris.

    (A) The behavioral response of Hydra to P3HT-NPs was evaluated using a contraction scoring system ranging from 6 (highly contracted) to 11 (elongated polyp). Animal behavior was monitored by continuous video recording for 8 min, according to the illumination protocol shown in the scheme, that is, 1-min dim light, 3-min white light illumination, 4-min dim light. Scale bars, 500 μm. (B) Average contraction scores and SD resulting from behavioral analysis (n = 60 polyps). Gray boxes indicate the 3-min light illumination period; black curves show contraction behavior of untreated polyps; red and blue curves show the contraction behavior of polyps treated with P3HT-NPs for 2 or 24 hours, respectively. SD values are reported as bars for each data set. (C) Average number of contraction events and relative SD estimated on treated (2 and 24 hours, red and blue lines, respectively) and untreated (black) polyps (n = 40, each condition). In most cases, statistically significant differences are observed (Student’s t test, *P < 0.05, **P < 0.01).

  • Fig. 3 P3HT-NP photoactivation enhances opsin3-like gene transcript levels.

    Hydra exposure to P3HT-NPs and/or white light illumination elicits a number of molecular reactions, which may provide useful clues on the transduction pathways activated by each stimulus. Genes selected in this study belong to light transductions pathway (opsin3-like), heat response (hsp70, trpa1-like), and oxidative stress (Cu-Zn SOD). Gene accession numbers for the Hydra homologous genes are reported in table S1. The expression profile of selected genes was investigated by qRT-PCR analysis using elongation factor 1α (HyEf-1α) as reference gene. Polyps treated with 0.25 μM P3HT-NPs for 24 hours underwent the illumination protocol shown on the upper part of the panel, then they were allowed to recover for either 30 min or 2 hours and were processed for RNA extraction and qRT-PCR analysis using specific primers (see table S1). opsin3-like gene expression shows great activation in response to both NPs and white light illumination, as compared to other conditions (untreated, not illuminated; treated, not illuminated). Data are presented as means ± SE of three technical repeats from two biological replicates. Statistical comparisons are performed using unpaired t test, *P < 0.05, **P < 0.01.

  • Fig. 4 Mechanisms underlying photoexcitation of P3HT-NPs.

    Top: Upon visible light illumination, primary photoexcitation states S1 are created within the polymer NPs, which can give rise to the creation of polaronic charged states P± or to nonradiative release of the excess energy ΔE. Bottom: different decay paths of the NP photoexcited states are shown: (A) photothermal excitation and heat release, (B) photoelectrochemical oxidation processes, and (C) electrical polarization.

  • Fig. 5 Biological pathways activated by P3HT-NP photostimulation.

    The sketch depicts a general epitheliomuscular ectodermal cell, presenting nucleus, mitochondria, and organelles. When light irradiates P3HT-NPs, both free and/or aggregated forms, electrons are generated in the photosensitive polymer, causing multiple biological responses. (A) NPs can localize within epitheliomuscular cells, and upon photoexcitation, a localized electrical dipole is established. In addition, photoelectrochemical reactions are promoted at the NP surface. These effects may locally act on neurons, leading to myofibril contraction and modulating the animal contracting behavior, as observed in behavioral studies. (B) Charged states sustain photoelectrochemical reactions, which increase the cytoplasmic concentration of ROS. This induces enzymatic reactions and acts on transcription factors (TF), or alternatively activates redox reactions of the respiratory chain and calcium binding transcription factors (CaM). In any case, targeted gene transcription is enhanced. (C) The retinal moiety of opsin3-like molecules or the P3HT-NPs themselves may initiate a light-dependent molecular cascade. GSH, glutathione.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/1/e1601699/DC1

    fig. S1. Characterization of P3HT-NPs.

    fig. S2. Impact of P3HT-NPs on Hydra population growth.

    fig. S3. Evaluation of polystyrene NP impact on Hydra behavior and gene expression.

    fig. S4. Light responsiveness of Hydra opsin3-like gene.

    fig. S5. Gene profiling under short illumination condition.

    fig. S6. Hydra opsin3-like gene transcription does not respond to heat stress.

    fig. S7. Alignment of opsin chromophore-binding transmembrane domain proteins from different animal species.

    fig. S8. Alignment of the predicted protein sequences representative of different classes of Hydra opsins.

    table S1. List of forward and reverse primers used in the qRT-PCR analysis.

    table S2. List of the opsin-like gene sequences present in the H. vulgaris genome database.

    References (5863)

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Characterization of P3HT-NPs.
    • fig. S2. Impact of P3HT-NPs on Hydra population growth.
    • fig. S3. Evaluation of polystyrene NP impact on Hydra behavior and gene expression.
    • fig. S4. Light responsiveness of Hydra opsin3-like gene.
    • fig. S5. Gene profiling under short illumination condition.
    • fig. S6. Hydra opsin3-like gene transcription does not respond to heat stress.
    • fig. S7. Alignment of opsin chromophore-binding transmembrane domain proteins from different animal species.
    • fig. S8. Alignment of the predicted protein sequences representative of different classes of Hydra opsins.
    • table S1. List of forward and reverse primers used in the qRT-PCR analysis.
    • table S2. List of the opsin-like gene sequences present in the H. vulgaris genome database.
    • References (58–63)

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