Experimental and Theoretical Studies of Novel Azo Benzene Functionalized Conjugated Polymers: In-vitro Antileishmanial Activity and Bioimaging

To study the effect of insertion of azobenzene moiety on the spectral, morphological and fluorescence properties of conventional conducting polymers, the present work reports ultrasound-assisted polymerization of azobenzene with aniline, 1-naphthylamine, luminol and o-phenylenediamine. The chemical structure and polymerization was established via Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (1H-NMR) spectroscopy, while the electronic properties were explored via ultraviolet-visible (UV-vis) spectroscopy. Theoretical IR and UV spectra were computed using DFT/B3LYP method with 6–311G basis set while theoretical 1H-NMR spectra was obtained by gauge independent atomic orbital (GIAO) method. The theoretically computed spectra were found to be in close agreement with the experimental findings confirming the chemical as well as electronic structure of the synthesized polymers. Morphology was investigated by X-ray diffraction and transmission electron microscopy studies. Fluorescence studies revealed emission ranging between 530–570 nm. The polymers also revealed high singlet oxygen (1O2) generation characteristics. In-vitro antileishmanial efficacy as well as live cell imaging investigations reflected the potential application of these polymers in the treatment of leishmaniasis and its diagnosis.


Synthesis of azo benzene based conjugated polymers.
Aniline (40 ml, 4.3 × 10 −1 mol) was added to an Erlenmeyer flask (250 ml) containing the synthesized diazonium salt (1.4057 g, 1 × 10 −2 mol). The reaction mixture was sonicated at 0-5 °C and after an interval of 15 min, ferric chloride (1 g, 3.8 × 10 −3 mol) was added to the above reaction mixture which changed from pale yellow to dark green. The reaction was further carried out for 5 h at the same temperature. The obtained polymer was then kept in a deep freezer for 24 h at −5 °C and was centrifuged, washed several times with distilled water on a R-8 C laboratory centrifuge and then dried in a vacuum oven for 24 h at 70 °C to ensure complete removal of solvent and other impurities. The synthesized polymers were designated as poly (aniline-azobenzene) (PANI-AB), poly(1-naphthylamine-azobenzene) (PNA-AB), poly (luminol-azobenzene) (PLu-AB), and poly (o-phenylenediamine-azobenzene) (PPd-AB) as shown in Scheme 1(a-e). The intrinsic viscosity was meaured as per method reported in our previous studies 23 . The values of intrinsic viscosity were calculated to be 0.41 for PANI-AB, 0.45 for PNA-AB, 0.42 for PLu-AB and 0.47 for PPd-AB. Hence, the weight average molecular weights (M w ) were presumed to be ranging between 10,500-19,000 27 . characterization Spectral analysis. FT-IR spectra of conjugated polymers were taken on FT-IR spectrophotometer (Shimadzu, Model IRA Affinity-1 while Ultraviolet-visible light (UV-vis) spectra were recorded on UV-vis spectrophotometer model Shimadzu, UV-1800 using CHCl 3 as solvent. 1 H-NMR spectra were recorded at 25 °C on a Bruker 300 MHz spectrometer using deuterated CHCl 3 . Fluorescence studies were performed on fluorescence spectrophotometer Fluorolog@ 3-11 using NMP solvent.
Growth reversibility and MTT assay. Leishmania donovani promastigotes untreated and treated with PANI-AB, PNA-AB, PLu-AB and PPd-AB (10 μg/ml) after 7 days were washed and resuspended in fresh media for the next 96 h. The parasites were fixed in 1% p-formaldehyde and counted on a hemocytometer using an inverted microscope. For the MTT assay, 2 × 10 5 THP-1 monocytic cell line per well were seeded onto a 96 well-plate and were treated with PMA for differentiation into macrophages for 24 h. Next day, the cells were washed, and the media was replaced with fresh media and were cultured for an www.nature.com/scientificreports www.nature.com/scientificreports/ additional 24 h in the presence of compounds PANI-AB, PNA-AB, PLu-AB and PPd-AB (100-6.25 μg/mL). Cell viability was determined using the MTT cell viability assay. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT (Sigma-Aldrich), was applied in the dark, following a 4 h incubation at 37 °C. The MTT-containing medium was replaced with 200 μL of isopropanol-HCl (0.1 N) and kept at 37 °C for 10 min to solubilize the formazan crystals. The samples were transferred to 96-well plates, and the absorbance of the converted dye was measured at 570 nm. The cell viability of the control (non-treated) cells was taken as 100%. In-vitro antileishmanial and cytotoxic activities were expressed as IC50 and CC50, respectively by the linear regression analysis.

Results and Discussion
Geometry optimization and distribution of charges as well as frontier molecular orbitals. The optimized structures of AB, PANI-AB, PNA-AB, PLu-AB and PPd-AB are given in, Fig. S1(a-e). The C-C and C=C bond lengths for AB molecule were noticed to be 1.39 Å and 1.38 Å respectively, whereas the C-N bond length was found to be 1.42 Å. The N=N bond length was found to be 1.07 Å. The bond lengths for optimized structure of PANI-AB were computed to be 1.40 Å and 1.35 Å respectively, while the C-N and N=N bond lengths were found to be 1.47 Å and 1.23 Å respectively. The N-H bond length was computed to be 0.998 Å. The structure was observed to be planar. Similarly, the optimized geometry of PNA-AB revealed the C-C and C=C bond lengths to be 1.42 Å and 1.35 Å respectively, while the C-N and azo bond lengths were observed to be 1.41 Å and 1.24 Å respectively. The N-H bond length was found to be 0.998 Å. The C-C and C=C bond lengths for PLu-AB were computed to be 1.39 Å and 1.35 Å respectively and the C-N, C=O, N-H and azo bond lengths were found to be 1.47 Å, 1.26 Å, 0.998 Å and 1.23 Å respectively. A slight reduction was noticed in the C-C and C=C bond lengths of this polymer as compared to the previous ones. The structure however was found to be planar. Interestingly, PPd-AB exhibited a highly twisted configuration and the C-C, C=C bond lengths were computed to be 1.38 Å and 1.37 Å respectively. The C-N bond length was observed to be 1.41 Å while the N-H bond length was computed to be 0.996 Å. The charge distribution and HOMO-LUMO orbitals in AB and its polymers are shown in Fig. 1(a-j). AB molecule showed charge concentration mainly around carbon atoms, Fig. 1(a), while in case of PANI-AB, the charge was found to be concentrated around the N-H bonds, Fig. 1(b). The charge distribution in PNA-AB, Fig. 1(c), and PPd-AB, Fig. 1(e), was found to be quite similar to that of PANI-AB whereas PLu-AB, Fig. 1(d), exhibited higher negative charge distribution on the carbon atoms adjacent to the C=O and NH linkages. The HOMO and LUMO orbitals were noticed to be highly delocalized in case of AB molecule, Fig. 1(f), as well as in AB modified polymers, Fig. 1(g-j) thereby confirming higher extent of spatial overlap between the two orbitals. This would result in the reduction of band gap, thereby facilitating charge injection of electrons into LUMO or holes into HOMO orbitals. Hence, hybridization of the conducting polymer chains upon insertion of AB was observed to reduce the band gap value as shown in Table 1. The reduction in HUMO-LUMO separation results in the formation of oligomers/polymers with near infrared (NIR) absorption characteristics. The HOMO-LUMO band gap for AB molecule was calculated to be 10.77 eV, Table 1, while it was found to be 5.50 eV for PANI-AB, 4.47 eV for PNA-AB, 3.89 eV for PLu-AB and 4.89 eV for PPd-AB. The band gap was found to be lowest for PLu-AB due to the presence of carbonyl linkages that facilitate the charge transfer through the polymeric chain.     It can therefore be concluded that the presence of the peaks associated with AB, PANI, PNA, PLu and PPd confirmed the polymerization as well as insertion azo benzene moiety. The presence of multiple NH stretching peaks, N 2 stretching peaks, CN vibration peaks as well as quinonoid/benzenoid peaks clearly confirmed the polymerization and successful insertion of AB in these polymers. The theoretical spectra of the polymers also revealed peaks in the same region thereby further confirming the proposed structures as well as insertion of AB in the polymers.
The 1 H-NMR spectrum of AB molecule (Given in Supplementary Information as Fig. S3(a)), Table 3 revealed peaks at δ = 7.27 ppm, 7.6 ppm and 8.2 ppm attributed to the aromatic protons of the benzene ring. Similar protons were also observed in the theoretical spectrum. The 1 H-NMR spectrum of PANI-AB (Given in Supplementary Information as Fig. S3(b)), Table 3, revealed a peak at δ = 4.4 ppm correlated to the protons of the amine group, while the proton associated with the NH linkage was observed at δ = 5.3 ppm. The theoretically computed 1 H-NMR spectrum of the same polymer showed protons of the amine group at δ = 4.51 ppm, 4.49 ppm while the protons of the NH linkage were noticed at δ = 5 ppm and 5.37 ppm. The protons associated with www.nature.com/scientificreports www.nature.com/scientificreports/ AB were noticed around δ = 7.3 ppm, 7.6 ppm and 7.8 ppm. The theoretical spectrum revealed the same protons around δ = 6.46 ppm and 6.56 ppm. The protons of the aniline ring were observed at δ = 6.8 ppm and were found to be in close agreement with the theoretical spectrum. Similarly, the 1 H-NMR spectrum of PNA-AB (Given in Supplementary Information as Fig. S3(c)), Table 3 showed protons of the amine group around δ = 4.7 ppm, while the theoretical spectrum revealed these protons at δ =4.35 ppm and 4.86 ppm. The protons associated with the benzene ring of 1-napthylmine were observed around δ = 6.7 ppm, 7.4 ppm and 7.8 ppm, while the protons of the azo benzene ring were observed at δ = 7.3 ppm and 7.5 ppm. The theoretical spectrum revealed these protons around δ = 6.53 ppm, 6.79 ppm, 6.81 ppm and 6.86 ppm. The small shift in the values was noticed due to the solvent effect. The 1 H-NMR spectrum of PLu-AB (Given in Supplementary Information as Fig. S3(d)), Table 3, also revealed protons of the amine group and the NH linkage at δ = 4.7 ppm and 5.6 ppm. The theoretical spectrum of the polymer revealed these protons around δ = 4.35 ppm, 4.86 ppm and 5.0 ppm, 5.4 ppm. The protons of the luminol ring were observed as a broad hump spanning between at δ = 7.4 ppm -7.9 ppm and the azo benzene protons were noticed at δ = 7.3 ppm. The broad hump obtained in the experimental spectrum was due to the presence of intense hydrogen bonding between the polymer chains. Similar hump was attained in case of PPd-AB (Given in Supplementary Information as Fig. S3(e)), Table 3. The presence of the NH protons as well as the amine protons confirmed the polymerization as well as insertion of AB in PANI, PNA, PLu and PPd. The theoretical spectrum was found to be closely matching with the experimental data which further established the formation of AB incorporated polymers.  www.nature.com/scientificreports www.nature.com/scientificreports/ intensity while a sharp peak was observed at 2θ = 25.1° corresponding to the plane typically observed in PANI as reported by other authors 29 . Interestingly, the XRD of PNA-AB exhibited several planes at 2θ = 10.60°, 11.61°, 17.07°, 18.28°, 19.4°, 21.3°, 22.52°, 23.73°, 26.56° and 29.2° confirming a highly crystalline and well organized structure. The rigidity and crystallinity were attributed to the presence of fused aromatic ring of PNA. The peak at 2θ = 21.1° appeared to be more pronounced and well formed. The XRD of PLu-AB exhibited an amorphous morphology as a single sharp peak was observed at 2θ = 27°. Similarly the XRD of PPd-AB revealed a broad diffuse hump around 2θ = 21.1° confirming the formation of an amorphous structure. It can therefore be concluded that the incorporation of AB in PANI, PNA, PLu and PPd showed structural reorganization and highly crystalline structure was attained in case of PNA-AB while amorphous structure was achieved in case of PPd-AB.

Morphological analysis via
The TEM of AB Fig. 2(a), showed the formation of a rod like structure containing tiny spherical particles. The TEM of PANI-AB, Fig. 2(b), exhibited flower like morphology with particle size ranging between 100-110 nm. The TEM of PNA-AB, Fig. 2(c), revealed dense spherical particles with sizes ranging between 20-25 nm, whereas the TEM of PLu-AB, Fig. 2(d), showed the formation of spherical clusters with particle size ranging between 25-45 nm. Simialrly, the TEM of PPd-AB, Fig. 2(e), showed clusters of tiny spherical nanoparticles ranging between 10-20 nm.

UV-visible, fluorescence emission and singlet oxygen generation kinetics. The experimental
and DFT/B3LYP calculated UV−vis spectra (shown in inset) are given in Fig. 3(a-e). The UV visible spectrum of AB, Fig. 3(a), showed a prominent peak at 285 nm while the theoretical spectrum revealed the same peak at 290 nm. Azobenzene can exist in E (trans) and Z (cis) forms which can be interconvertible via photochemical as well as thermal processes 30 . Since the Z-E energy difference is approximately in the range 47-48 kJ mol −1 , the thermodynamically stable form is the E isomer of AB and the conversion of cis isomer to trans isomer is generally irreversible under ambient conditions 30 . The UV spectrum obtained for AB molecule in our case was similar to the one reported for the trans form 31 . Interestingly, the peak corresponding to the existence of trans isomer of AB molecule was found to be pronounced in all the modified polymers which confirmed that upon polymerization, AB existed in its most stable form (trans isomer). The peaks in the UV region noticed in the experimental as well as thoeretical spectra were correlated to the π−π* transition of the benzenoid ring and the oscillator strength values were calculated to be 0.25, 0.26 respectively, Table 1. The UV spectrum of PANI-AB, Fig. 3(b), showed peaks at 310 nm and 400 nm. The later peak was associated with the n−π* transition which was also noted in the theoretical spectrum. The oscillator strength was computed to be 0.83 and 0.85 for the experimental and theoretical peaks respectively. Similarly, the UV spectrum of PNA-AB showed peaks at 280 nm 450 nm and the theoretical spectrum showed a pronounced peak at 460 nm. The oscillator strength values were similar to those observed in PANI-AB and could be correlated to the structural similarity of the two polymers. The polymer PLu-AB, Fig. 3(d), exhibited a prominent peak at 280 nm while a broad hump corresponding to n−π* transition was observed around 520 nm. Likewise, the spectrum of PPd-AB showed an extensive peak around 370 nm which was observed at 400 nm in the theoretical spectrum, Fig. 3(e). The difference of around 30 nm observed in the theoretical and experiment spectrum could be correlated to solvent effect as PPd shows intesne hydrgen bonding with polar solvents. The experimental and theoretical spectra were observed to be in good agreement which confirmed the proposed chmical structure of the polymers. www.nature.com/scientificreports www.nature.com/scientificreports/ Upon excitation at 480 nm, the fluorescence spectra of PANI-AB and PPd-AB exhibited emission maxima at 570 nm while PNA-AB and PLu-AB showed broad emission humps spanning between 500-570 nm corresponding to S 1 →S o transition 22,27 , Fig. 4(a). The quantum yield value was observed to be highest for PPd-AB while it was noticed to be lowest for PANI-AB, Table 1. Upon light irradiation, the polymers were observed to undergo transfer to the excited state and interacted with dissolved oxygen to generate 1 O 2 . The 1 O 2 generation studies were therefore carried out using diphenyl isobenzofuran (DPBF) as quencher 32 . The assays were performed by dissolving the polymers in chloroform (20 µg/mL) and DPBF (6 × 10 −5 M). The UV spectra were taken upto 50 secs at regular intervals of 10 secs under continuous excitation of laser light (650 nm). The UV absorbance of DPBF at ~410 nm was plotted as a function of the irradiation time (given in Supplementary Information as Fig. S5(a-d)). The studies showed that pure DPBF showed negligible changes under the laser light irradiation. The polymers however showed a prominent decrease in the absorbance value at ~410 nm. A plot of optical absorbance at 410 nm as a function of irradiation time was consistent with first order kinetics, Fig. 4(b). The kinetic plot revealed that PPd-AB, Fig. 4(b), exhibited the highest k value of 9.1 × 10 −4 s −1 while PANI-AB showed a value of 3.2 × 10 −4 s −1 . The 1 O 2 generation quantum yield values for the polymers were determined by reported method 32 . The 1 O 2 quantum yield value for the PPd-AB was found to be 0.091. It can therefore be concluded that the polymers can act as effective photosensitizers and can be utilized in photodynamic therapy.
Studies on antileishmanial activity. Leishmania evades immune host immune system by dampening critical signalling pathways which are required for parasite clearance 5,6 . In this regard, the development of less toxic nanoparticles could also be helpful in providing therapeutic benefits 7 . The potential antileishmanial activity of PANI-AB, PNA-AB, PLu-AB and PPd-AB was explored and the polymers were found to be non-toxic as compared to miltefosine, the standard antileishmanial drug at equivalent concentrations. The polymers showed in-vitro antileishmanial activity up to 48 h, Fig. 5(a). The IC 50 value for miltefosine is 6.9 µg/ml. The IC 50 values of PANI-AB, PLu-AB, PNA-AB and PPd-AB were evaluated as 10.7 µg/ml, 23.9 µg/ml, 12.3 µg/ml, 19.2 µg/ml respectively. These polymers were further tested for promastigote growth kinetics and both PANI-AB as well as PNA-AB were found to be more effective against promastigotes in comparision to PPd-AB. PLu-AB was the least effective among all the tested polymers, Fig. 5(b). The cytotoxicity assays on THP-1 cell line derived, human macrophages showed that the polymers could be safely used at a concentration of 10 µg/ml as compared to miltefosine, Fig. 6(c). Furthermore, the parasites were washed after the treatment and monitored for growth reversibility, and it was found that promastigotes treated with PANI-AB, PNA-AB and PPd-AB failed to return to a viable state, while untreated promastigote returned back to late log phase. However, PLu-AB treated promastigotes did not revert back to normal morphology after 96 h, Fig. 5(d). The possibility of using these fluorescent polymers as effective imaging agents for Leishmania parasites was investigated by carrying out live cell imaging experiments by treating Leishmania parasites with the polymers as depicted in Fig. 6(a-d).
It was observed that PANI-AB, Fig. 6(a), exhibited intense green and red emission as compared to blue emission while the emission intensity of PLU-AB, Fig. 6(b), was observed to be lower. This was due to higher extent of penetration of PANI-AB into the promastigote as compared to PLu-AB. Similarly, in case of PPd-AB, Fig. 6(c), the intensity of green emission was observed to be higher as compared to blue emission while for PNA-AB, Fig. 6(d), the extent of penetration of the polymer was found to be higher under different lasers. This was further confirmed by the 3D image, Fig. 6(e). The images clearly revealed that the polymer binds to the plasma membrane before trafficking via endocytic pathway. The polymers can therefore be effectively used to stain and image Leishmania promastigotes to monitor their growth as well as apoptosis.
conclusion Azo modified polymers were synthesized and confirmed by experimental as well as theoretical FTIR, UV-visible and 1 H-NMR studies. The polymers exhibited intense fluorescence in the IR region and the intensity of emission was governed by the extent of conjugation attained. Antileishmanial activities showed that the polymers PANI-AB, PNA-AB, and PPd-AB have potent in-vitro antileishmanial activity. Cytotoxicity studies on human macrophages revealed that the polymers were potentially safe at doses equivalent to miltefosine. The live cell imaging studies of PNA-AB stained Leishmania promastigotes showed intense emission in green as well as red regions. As these polymers were found to be least-toxic, they can be safely used as fluorescent markers to label and target parasite in Leishmania infected patients.