Influence of Luminol Doping of Poly(o-phenylenediamine) on the

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Influence of Luminol doping of poly(o-phenylenediamine) on the spectral, morphological and fluorescent properties: A potential biomarker for Leishmania parasite Ufana Riaz, Sapana Jadoun, Prabhat Kumar, Mohd. Arish, Abdur Rub, and Syed Marghoob Ashraf ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10325 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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ACS Applied Materials & Interfaces

Influence of Luminol Doping of Poly(ophenylenediamine) on the Spectral, Morphological and Fluorescent properties: A Potential Biomarker for Leishmania Parasite Ufana Riaza*, Sapana Jadouna, Prabhat Kumarc, Mohd.Arishb, Abdur Rubb,d, and S.M.Ashrafa† a

Materials Research Laboratory Department of Chemistry, Jamia Millia Islamia,New Delhi110025, India, b Department of Biotechnology, Jamia Millia Islamia,New Delhi-110025

c

Advanced Instrumentation Research Facility, Jawaharlal Nehru University, New Delhi- 110067

d

Department of Medical Laboratory Sciences, College of Applied Medical Science, Majmaah

University, Al Majmaah, Kingdom of Saudia Arabia (KSA) *

Corresponding author email: [email protected], † now retired

KEYWORDS: doping; conjugated polymer; fluorescence; band gap; cytotoxicity; bioimaging ABSTRACT

There has been a steady progress in the development of doped conjugated polymers to remarkably improve their photo physical properties for their application as biomarkers. With a

ACS Paragon Plus Environment

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view to enhance the spectral, morphological and photo physical properties of poly(ophenylenediamine) (POPD), the present work reports the synthesis of poly(o-phenylenediamine) and doping of this polymer using luminol. The formation of luminol doped POPD was confirmed by infrared, ultraviolet-visible (UV-vis) spectroscopies and X-ray diffraction (XRD) studies. The energy band gap values and oscillator strength of luminol in acidic, basic, and neutral media were computed by density functional theory (DFT) calculations using the B3LYP/6-31G (d) basis set and were compared with experimental data. The cell viability was investigated using the methyl tetrazolium (MTT) assay while in-vitro anti-leishmanial activity was determined using inhibitory concentration (IC50) and cytotoxic concentration (CC50) respectively. The results revealed that luminol doped POPDs were on potentially non cytotoxic and exhibited immense potential to be used as a biomarker for Leishmania donovani.

INTRODUCTION The identification and characterization of intracellular events is of immense importance particularly for therapeutic applications1-5. Optical analysis based on fluorescence labeling has been extensively used to study various biological mechanisms and quantum dots such as cadmium selenide (CdSe) cadmium telluride (CdTe), clusters of silver and gold nanoparticles as well as mesoporous silica nanoparticles are most often used in these cases4-7. Although these fluorophores have high quantum yield, they usually suffer from photobleaching and present a certain level of toxicity that hinders their application in bioimaging8. Lately, there has been steady progress in the development of highly fluorescent conjugated polymers such as polyaniline (PANI) 9, polypyrrole (Ppy) 10, poly(1-naphthylamine) (PNA)11 that can be encapsulated and functionalized by doping12. Among the several processible PANI


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derivatives, poly(o-phenylenediamine) (POPD) is known to possess high aromaticity due to the presence of quinoxaline repeating units13. It exhibits strong adsorbability and has been utilized for designing fluorescence probes, chemical and biological sensors14-15. Doping is one of the facile techniques that has been extensively utilized to alter the structural properties of materials and the use of conjugated polymers confers useful advantages, such as the introduction of functionality and charge carriers on the surface of these polymers16-17. Enhancement in the physico-chemical properties can be easily achieved through the tuning of band gap of these polymers by the introduction of dopants18-19. Dye-doped polymer systems show fluorescent effects by using high concentrations of the active molecules while preventing the aggregation of the molecules in the polymer matrix20. Non-covalent intermolecular interactions help in binding functional molecules to the polymer backbone and are widely exploited in supramolecular chemistry to prepare self-assembled polymeric materials. Although the influence of dye-dye intermolecular interactions on the performance of polymer systems have been thoroughly investigated, less effort has been directed towards the study of the effect of covalent interactions between other functionalized active molecules on the conjugated polymer as host material18-20. Luminol (5-Amino-2,3-dihydro-1,4 phthalazinedione) is a well-known chemiluminescent reagent vastly used in molecular biology and analytical chemistry. It has been used as the basis for a multitude of sensitive and selective detection methods including high performance liquid chromatography (HPLC), immunoassay, DNA probes, DNA typing and as substrate in western blot detection21-25. Due to the relevance of some of its chemiluminescent applications, several groups have searched for some luminol derivatives to maximize the chemiluminescence quantum yield of this compound and extend the range of emission wavelengths towards the visible region


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of the electromagnetic spectrum26-29. Even though some of the experiments have been quite successful, the study of this phenomenon is far from being exhausted, because the variable oxidative states of luminol in acidic and basic media are not completely established. The oxidation reactions of luminol may proceed in several ways depending on the pH of the medium18. In this paper, we have attempted to investigate the influence of covalent interactions between luminol (Lum) and POPD in acidic, basic as well as neutral media. The systems were studied by ultraviolet-visible (UV-Vis) and infrared (IR) spectroscopy, X-ray diffraction measurements and transmission electron microscopic techniques. The results showed that intermolecular interactions between luminol and POPD could be enhanced by changing the oxidation state of the former. The aggregation tendency was observed to be minimized and luminol doping considerable enhanced the fluorescence emission of POPD thereby providing a new method to synthesize tailor made functional polymers with no need for complex organic synthesis. This could open new avenues for designing materials suitable for various biological and optoelectronic applications. Leishmania donovani stain was chosen for investigating the cytotoxicity and in-vitro anti-leshmanial activity. Leishmaniasis is a vector-borne disease caused by a protozoan that can lead to lethal visceral leishmaniasis and has infected about 12 million people worldwide30. Till date, the only control intervention chemotherapy against leishmaniasis involves the use of highly toxic pentavalent antimonials, for which resistance has been extensively reported31. Hence the use of luminol POPDs as biomarker is expected to help in the early detection and diagnosis of this parasite. The cell viability was determined using the MTT cell viability assay while in-vitro anti-leishmanial activity was expressed as inhibitory concentration value (IC50) and cytotoxic concentration values (CC50) respectively, by linear regression analysis.


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MATERIALS AND METHODS o-phenylenediamine (Sigma Aldrich, USA), luminol (Sigma Aldrich, USA), ferric chloride (Merck, India), Sodium hydroxide (NaOH) (Merck, India), tetrahydrofuran (THF) (Sigma Aldrich, USA), N-Methyl-2-pyrrolidone (NMP) (Sigma Aldrich, USA), Hydrogen peroxide (H2O2) (50 wt. % in H2O, stabilized) (Sigma Aldrich, USA), Potassium ferrocyanide (K4Fe(CN)6) (Merck, India) were without further purification. Ultrasonic synthesis of poly(o-phenylenediamine) (POPD) O-phenylenediamine monomer (4 g, 0.037 mol) was added to 250 ml Erlenmeyer flask containing deionized water (100 ml). The solution was stirred at room temperature for 15 min. Ferric chloride (5.99 g, 0.036) used as initiator dissolved in distilled water (150 ml) was added to reaction mixture drop by drop with the help of burette keeping the monomer: initiator ratio as 1:1. The flask was then sonicated for 3 h, maintained between 25-30 οC using an ultrasonicator. The color of reaction mixture changed from transparent brown to greenish brown indicating polymerization of o-phenylenediamine11. The same was kept in a deep freezer for 24 h at -5 οC. The synthesized polymer was washed several times with distilled water with the help of R-8C laboratory centrifuge to confirm the removal of excess ferric chloride which was tested with potassium ferrocyanide. Poly(ophenylenediamine) was then dried in a vacuum oven at 100 oC for 72 h to ensure complete removal of unreacted monomer, impurities, and

water. The polymer was

designated as POPD and the percentage yield was calculated to be 89.81 %. Preparation of Luminol doped POPD polymers


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Solutions of luminol were prepared in neutral, acidic and basic media by dissolving luminol (2.1 g) in deionized water (250 ml), 0.1 M sodium hydroxide (NaOH) solution (250 ml) and 0.1 M of hydrogen peroxide (H2O2) solution (50 wt. % in H2O) (250 ml). Finely grinded POPD powder (500 mg) was then dispersed in the above luminol solutions separately and were subjected to sonication for a period of 8 h at 25⁰C. The solutions were kept undisturbed overnight. The doped polymers were then separated from the solutions via centrifugation and dried in a vacuum oven for 72 h at 100 oC to ensure complete removal of water and other impurities. The solutions of luminol prepared in neutral, acidic and basic media were designated as Lum-H2O, Lum-H2O2 and Lum-NaOH respectively while the doped polymers were designated as POPD-Lum-H2O, POPD-Lum-H2O2 and POPD-LumNaOH, respectively. CHARACTERIZATION DFT and TD-DFT calculations were performed using GAUSSIAN 09 software; the results were analyzed with Gauss View software. The optimized geometries of luminol in different media were determined by gradient minimization at DFT with correlation functions B3LYP/ at 6-31G (d) basis set by ignoring symmetries. The geometry optimizations were considered complete when a stationary point was located on the Potential Energy Surface (PES). The UV−vis spectra of optimized geometric structures of luminol in neutral, acidic and basic solutions were simulated at the TD-DFT/B3LY, UB3LYP-6- 31+G(d, p) level. The frontier molecular orbital simulations such as energies of highest occupied molecular orbitals (HOMO), energies of lowest unoccupied molecular orbitals (LUMO) and band gap calculations were performed using DFT/B3LY/6-31G level. Viscosity average molecular weight was calculated by taking the Mark-Houwink constants (K and a), as first approximation, where [K = 2.0 x 10-2 and a =


6

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0.3176] (0.2 wt %) in NMP medium using Ubbelhode Viscometer, as reported in our earlier studies18-19. FT-IR spectra of polymers and doped polymers were taken in KBr pellets on FT-IR spectrometer model Shimadzu IRA Affinity-1. The integrated absorption coefficient, ∫ adν , was determined using the IRA Affinity-1 software through Gaussian Lorentzian curve fittings as shown in Table.1. UV-visible spectra were taken on UVvisible spectrophotometer model Shimadzu UV-1800 using NMP as solvent. The molar extinction coefficient and oscillator strength were calculated as reported in our earlier studies32. X-ray diffraction patterns of the polymers and doped polymers were recorded on Philips PW 3710 powder diffractometer (Nickel filtered copper K-α radiation). The d spacing (D) was determined using Bragg’s relation32. High Resolution Transmission electron micrographs (HRTEM) were taken on TECNAI 200 Kv TEM (Fei, Electron Optics). Fluorescence emission spectra were recorded on fluorescence spectrophotometer Fluorolog@3-11 in solution medium using NMP as solvent. The quantum yield (ϕ) was calculated was calculated using the Rhodamine B as reference material as mentioned in our previous work11. Confocal micrographs were obtained using a using a Laser Confocal Microscope with Fluorescence Correlation Spectroscopy (FCS) - Olympus FluoView™ FV1000 equipped with He-Ne laser and oil immersion objective. λmax for laser excitation was 410 nm. Cell Culture and anti-leishmania activity: Dose-dependent evaluation of anti-promastigote activity and determination of IC50 Leishmania donovani (MHOM/IN/1980/AG83) promasigotes were grown in M199 medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco) and 1% Penicillin streptomycin solution (Gibco) at 22°C. Promastigotes at a density of 2 × 106 cells/ml


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were incubated in the absence and or presence of compounds 1-4 at serial dilutions starting at 100 µg/ml (100, 50, 25, 12.5, 6.25, 3.125 µg/ml) for 48 h at 22°C. Miltefosine hydrate was used as a standard anti-leishmanial drug. THP-1 cells were maintained in a 5% CO2 humidified incubator at 37°C in RPMI medium (Gibco) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco) and 1% Penicillin streptomycin solution (Gibco). THP-1 cells were differentiated using phorbol 12-myristate 13-acetate (PMA) at 5ng/ml. Dose-dependent evaluation of anti-promastigote activity and determination of IC50 promastigotes at a density of 2 × 106 cells/ml were incubated in the absence and or presence of luminol doped POPDs at serial dilutions starting at 100 µg/ml (100, 50, 25, 12.5, 6.25, 3.125 µg/ml) for 48 h at 22°C. Miltefosine hydrate was used as a standard anti-leishmanial drug. Cells were counted using hemetocytometer. For MTT assay, 1×105 cells per well were seeded onto 96-wells plate and was treated with PMA for differentiation for 24 h. Next day cells were washed and media was replaced with fresh media and cultured for additional 24 h in presence of compounds 1-4 (100031.25 µg/ml). Cell viability was determined using the MTT cell viability assay. 3-(4,5-Dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT (Sigma–Aldrich) was applied at in dark following a 4 h incubation at 37°C. The MTT containing medium was replaced with 100 µl of isopropanol-HCl (0.1N) 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 percent cell viability of the control (non-treated) cells was taken as 100%. In-vitro anti-leishmanial and cytotoxicity activity was expressed as IC50 and CC50 respectively, by linear regression analysis. Values are mean ± SD of samples in triplicate from two independent experiments.


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RESULTS AND DISCUSSION Theoretical determination of chemical structures of luminol in acidic, basic and neutral media. The optimized structures and charge distribution of luminol structures in acidic, basic, and neutral media are given in supporting information, Figures S1 and S2 and Tables S1, S2 and S3. The optimized structures as well as the corresponding Cartesian coordinates of the Lum-H2O given in Figure S1 (a), Table S1 reveal that the C=C and C-C bond lengths were noticed to be 1.33 Å and 1.51 Å respectively, while the C=O bond length was found to be in the range of 1.24 Å − 1.27 Å. The C−N bond length was found to be 1.42 Å and the N-N bond length was calculated to be 1.41 Å. The optimized structures as well as the corresponding cartesian coordinates of the Lum-H2O2, Figure S1 (b), Table S2 showed that the C=C and C-C bond lengths were noticed to be 1.45 Å and 1.47 Å respectively, while the C-O and C=O bond lengths were found to be 1.41 Å and 1.25 Å. The C−N bond length was found to be 1.50 Å. The optimized structures as well as the corresponding cartesian coordinates of the Lum-NaOH, Figure S1 (c), Table S3 revealed that C=C and C-C bond lengths were noticed to be 1.39 Å and 1.48 Å respectively whereas the C−N bond length was found to be 1.48 Å and the N=N bond length was noticed to be 1.27 Å. The C=O bond length was observed to be 1.27 Å. Analysis of the optimized parameters indicates that geometrical distortion takes place while moving from neutral to basic species, because the C−N bond length revealed slight variation which increases by about 0.08 Å for the acidic luminol and 0.06 Å for basic luminol. Similar trend was observed in the variation of the dihedral angles which could be correlated to the existence of various forms of luminol in neutral, acidic and basic media. In neutral medium, luminol is found to exist in


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fully protonated form, Figure S1(a), while in acidic medium it undergoes oxidation to produce 3aminophthalate, Figure S2(b). The basic form leads to the formation of a 5 aminophthalazine 1, 4-dione , Figure S1(c). The distribution of the charges in structures of luminol obtained under different media is shown in Figure S2. Lum-H2O ,Figure S2(b), exhibited higher negative charge distribution on the carbon atoms of the aniline ring while the carbons atoms linked to the oxygen showed lowest charge. The nitrogen atom of the aniline ring was observed to be more electronegative than the nitrogen atoms that were adjacent to the carbonyl group. The charge distribution incase of LumH2O2, Figure 2(b), was observed to be quite different. The oxygen atoms adjacent to the carbonyl groups were noticed to be less electronegative as compared to the oxygen of the carbonyl groups. The nitrogen atom of the aniline ring as well as the carbon atoms located at the meta and para positions with respect to the amino group were noticed to bear higher negative charge whereas for Lum-NaOH, Figure 2(c), the nitrogen atoms that were adjacent to the carbonyl group were found to bear negative charges similar to the oxygen atoms present in the same position in case of Lum-H2O2, Figure 2(b). It can thus be concluded that the charge distribution was found to vary for luminol doped in different media. The DFT/B3LYP calculated UV−vis spectra of the three forms of luminol are given in Figure S3 of supporting information. The experimental UV visible spectra of luminol ,Figure1, revealed 3 peaks at 260 nm, 350 nm and 410 nm respectively. The first two peaks were correlated to the π−π* transition of the benzenoid while the third peak was related to the n−π* transition. The intensity of the peak corresponding to n−π* transition was observed to be highest for Lum-NaOH while the peak intensities of Lum-H2O and Lum-H2O2 were observed to be close to each other.


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Figure 1 UV-visible spectra of luminol in acidic, basic and neutral luminol solutions

The simulated UV−vis spectra revealed a peak around 420 nm in neutral, acidic as well as basic media and matched well with the values reported by other authors for luminol33. The later peak was closely related to the experimentally determined peak in our case and the simulated spectra could be correlated to the proposed structures. The frontier molecular orbital simulations at DFTB3LYP/6-31G level of theory yield energies of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of neutral, acidic, and basic forms of luminol. The HOMO−LUMO orbitals for neutral, acidic and basic structures are depicted in Figure S4 in the Supporting Information. The HOMO−LUMO energy band gap was calculated to be 0.087 eV, 0.043 eV, and 0.40 eV, respectively for neutral, acidic and basic luminol structures, Table 1. The band gap was observed to be higher in neutral media while in acidic and basic media, the band gap values were noticed to be almost similar which could also be correlated to the charged


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distribution structures of luminol, Figure S2. The oscillator strength values determined by the experimental data as well as by the simulated spectra were also observed to be closely matching. Table 1 HOMOs, LUMOs, and band gap energies of luminol in neutral, acidic and basic media Species

HOMO LUMO

Bandgap Oscillator strength

(eV)

(eV)

(eV)

Calculated

Theoretical

λmax (400 nm) λmax(400 nm) Lum-H2O

-0.2721 -0.1847 0.087

0.0052

0.0060

Luminol-H2O2

-0.2586 -0.2160 0.043

0.1500

0.1200

Luminol-NaOH -0.2707 -0.2303 0.040

0.0530

0.0580

Intrinsic viscosity and molarity of luminol doped POPD polymers To estimate the molecular weight of synthesized polymers, dilute solution viscosity method was used and with the help of intrinsic viscosity, viscosity average molecular weight measured by using Mark-Houwink equation: [η] = 2.0 × 10-2 Mv0.3176 taking PANI as reference ,Table 2. Pure POPD showed an increase in intrinsic viscosity upon doping. Among all the doped polymers, highest viscosity was observed for POPD-LumH2O while lowest viscosity was shown by POPD-Lum-NaOH. The increase in the viscosities and the viscosity average molar masses confirmed the doping of POPD by luminol.


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Table 2 Intrinsic viscosity and molar mass of POPD and luminol Doped POPD Sample

C%

H%

N%

Intrinsic

Viscosity

Empirical

Molecular

Viscosity

average molar

formula

formula

mass

(η)

(Mv) POPD

46.59 4.95 16.47 0.37

8922

C3.29H4.20N

C272H347N82

POPD-Lum-H2O

64.16 4.55 22.31 0.49

21528

C3.35H2.85N

C667H568N199

POPD -Lum-H2O2

62.43 4.46 21.83 0.47

18906

C3.33H2.85N

C583H499N174

POPD-Lum-NaOH

51.54 3.57 17.43 0.41

12328

C3.44H2.86N

C393H326N114

Confirmation of doping of POPD by luminol via UV-visible and FTIR analyses The UV-Visible spectrum of POPD and luminol doped POPD in THF revealed peaks at 256 nm and at 420 nm, Figure 2. The peak at 256 nm was associated with the π-π* transition of benzenoid rings. The peak at 420 nm was associated with the n-π* transition The oscillator strength for this peak was observed to be 0.14. Upon doping of POPD with luminol in various media, the intensity of the peak associated with polaronic transition of POPD revealed an increase in the intensity which conformed doping of POPD by luminol 18-19

. The oscillator strength was calculated to be 0.26 for POPD-Lum-H2O, while it was

found to be 0.25 for POPD-Lum-NaOH. POPD-Lum-H2O2 showed an oscillator strength


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value of 0.19, Table 3. The higher values of oscillator strength confirmed higher transition ability of electrons in the doped polymers as compared to pure POPD.

Absorbance

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POPD POPD-Lum-NaOH POPD -Lum-H2O2

3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2

POPD- Lum-H20

210 240 270 300 330 360 390 420 450 480 510 540 570 600

Wavelength (nm)

Figure 2 UV-visible spectra of POPD and Luminol doped POPD in acidic, basic and neutral media Table 3 UV data of luminol and luminol doped POPD Sample

pH

λmax(nm) ∫adν̅ (cm−2)

Oscillator strength

POPD

-

418

907.94

0.14

POPD- Lum-H2O

7.30

414

1700.00

0.26

POPD-Lum-NaOH

10.95

415

1619.36

0.25

POPD -Lum-H2O2

3.12

414

1244.63

0.19


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The data of IR spectrum of pure POPD ,Table 4, revealed an N-H stretching vibration peak at 3418 cm-1 due to the presence of secondary amine (-NH-)18-19. The area under the peak was calculated to be 610 cm-2. The peaks at 1573 cm-1 and 1381 cm-1 were assigned to ring puckering of the quinonoid and benzenoid rings respectively and the area of quinonoid peak was more than benzenoid and B/Q ratio was found 0.15. The CN stretching vibration peak was observed at 1240 cm-1. The peak at 865 cm-1 was associated with para-substituted benzene while the peak at 744 cm-1 was the characteristic peak of CH out-of plane bending vibrations present on benzene nuclei in the phenazene skeleton. The presence of above peaks confirmed the polymerisation of POPD11,18-19. Upon doping of POPD with Lum-NaOH, the NH streching peak was found to shift from 3418 cm-1 to 3541 cm-1 while integrated area under the NH peak increased up to 967 cm-2 which indicated doping of POPD by luminol. The imine stretching peak appeared at 1643 cm-1 which was absent in pristine POPD. The quinonoid and benzenoid peaks were found to shift from 1573 cm-1 and 1381 cm-1 observed in case of pure POPD to 1505 cm-1 and 1350 cm-1. The B/Q ratio in this case was found to be 0.70 confirming the formation of higher number of benzenoid units. The integrated area of CN stretching peak at 1233 cm-1 also increased indicating the doping of POPD by luminol. The para-substituted benzene and phenazene skeleton peaks were also observed to shift from 865 cm-1 and 744 cm-1 to 880 cm-1 and 771 cm-1. Likewise in POPD-Lum-H2O2, the NH stretching vibrational peak was observed at 3428 cm-1 which revealed a shift of about 10 cm-1 as compared to pristine POPD and area under NH peak was found to be 902 cm-2. In POPD-Lum-H2O, the NH peak was noticed at 3450 cm-1 with the ∫adῡ being 1081 cm-2 which was a noticeable increase showing the doping of POPD in neutral medium while the peak at 1658 cm-1


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corresponding to imine stretching and the ratio of benzenoid and quinonoid peak is 1.05 showing more benzenoid units in structure. Table 4 FTIR spectral data of POPD and luminol doped POPD in acidic ,basic and neutral media

Sample

Functional Group

Peak

Absorption

∫adῡ

position Intensity (cm-1) POPD

NH- stretching

3418

1.61

610

C-C stretching (quinonoid) 1573

1.28

209

C-C stretching (benzenoid) 1381

0.72

32

CN stretching (benzenoid)

1240

0.69

29

p-substituted benzene

864

0.56

37

Phenazine skeleton

744

0.57

25

3541

0.79

967

1643

0.68

59


0.67

78


0.62

55


1233

0.60

90

p-substituted benzene

880

0.52

38

Phenazine skeleton

771

0.56

41

NH- stretching

3428

2.33

902


1.55

446

Phenazine skeleton

799

1.06

94

NH- stretching

3450

1.46

1081

POPD- Lum-NaOH NH- stretching Imine stretching

POPD -Lum-H2O2

POPD- Lum-H2O


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Imine stretching

1658

0.97

143


0.93

73


0.80

77


1220

0.71

92

Phenazine skeleton

777

0.62

42

Nature of crystallinity and morphology of POPD and Luminol doped POPD XRD of pristine POPD, Figure 3 (inset), revealed three intense peaks at 2θ = 9.14o, 10.58o, 18.13o corresponding to (010), (200) and (110) planes. The sharpness of the peaks reflected the well-ordered arrangement of the POPD chains along different planes11,18-19. Among the other planes, the sharpness as well as the peak area of (110) plane was the highest having an inter-chain distance of 6.1 A⁰. Upon doping of POPD with luminol shifting of the peaks was observed. POPD-Lum-H2O revealed four intense peaks at 2θ = 10.07 o, 12.60o, 15.11o, and 15.96o. The intensity of the peak around 18o was found to appreciably decrease. POPD-Lum-NaOH and POPD-Lum-H2O2 also revealed similar peaks but the peak intensities were observed to be lower than that of POPD-Lum-H2O2.


17


160000

POPD-Lum-NaOH POPD -Lum-H2O2 POPD- Lum-H2O

70000

POPD

60000 50000

Intensity

140000 120000

40000 30000

100000

Intensity

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20000 10000

80000 0 6

8

10

12

14

16

18

20

2-theta

60000 40000 20000 0 6

8

10

12

14

16

18

20

2-Theta

Figure 3 XRD of POPD and luminol doped POPD in acidic, basic and neutral media The area under the peak noticed at 15.11o was observed to be highest for POPD-Lum-H2O a d lowest for POPD-Lum-NaOH. The increase in the peak area could be correlated to the increase in the lattice strain produced upon doping of POPD with luminol (Table S4). The presence of well-formed peaks reflected the crystalline nature of POPD which was found to remain intact even after doping. The slight shifting of the peaks as well as the increase in their intensities clearly confirmed doping with luminol. The HRTEM of POPD, Figure 4 (a), revealed mixed morphology of needle and distorted spherical aggregates. The morphology of POPD-Lum-H2O, Figure 4(b), exhibited the formation of fused hollow tubes consisting of dense and light particles while the morphology of POPDLum-H2O2, Figure 4(c), exhibited the formation of core shell spherical particles. Small particles were seen to surround a huge distorted spherical agglomerate which resemble the one observed


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for pure POPD. It appeared as if tiny spherical particles of luminol surrounded the sense POPD cluster. Likewise, the HRTEM of POPD-Lum-NaOH, Figure 4(d), showed the formation of a fused core shell like structure in which a thin layer of luminol was noticed to surround the dense distorted spherical particle of POPD core. The morphologies could be well correlated to the observations made in our previous studies thereby confirming the doping of POPD with luminol.

(a)

(b)

(c)

(d)

\

(c)

(d)

Figure 4 TEM of of (a) POPD, (b) POPD-Lum-H2O,(c) POPD-Lum-H2O2 ,(d) POPD-LumNaOH


19


Fluorescence Studies of POPD and luminol doped POPD

POPD POPD-Lum-NaOH POPD-Lum-H2O2 POPD-Lum-H2O

5000

4000

Intensity (CPS)

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3000

2000

1000

0 0

2

4

6

8

Time (ns)

Figure 5 Fluorescence spectra of POPD and luminol doped POPD in acidic, basic and neutral media, (inset shows the time resolved spectra of POPD and luminol doped POPD)

The emission spectrum of POPD and luminol doped POPD in THF were obtained upon excitation at of the solutions at 500 nm and are depicted in Figure 5. The emission spectrum of POPD revealed a peak at 620 nm corresponding to S1→S0 transition. The intensity of this transition was found to be influenced by the nature of the dopant. The emission spectrum of POPD-Lum-H2O , Figure 5, showed a slight blue shift and was found at 613 nm. The intensity of the peak was noticed to be highest (14000 CPS) among all the other doped POPDs. Likewise, the fluorescence emission peak of POPD-Lum-NaOH also revealed a blue shift towards 618 nm but the intensity was observed to be lower than the previous case. POPD-


20

10

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Lum-H2O2 revealed a blue shift at 613 nm with intensity upto 890 CPS which was lowest among all doped POPDs. The intensities of fluorescence emission were found to be higher in all the three cases as compared to pristine POPD. The values of quantum yield (ɸ) values were calculated to be 0.13 for POPD, 0.19 for POPD-Lum-H2O, 0.18 for POPD-Lum-H2O2, 0.16 for POPD-Lum-NaOH. The fluorescence decay time (τ), was found to be was inversely proportional to the Φ, i.e., lower the decay time, higher the quantum yield. The decay time was determined from the curves by reading off the time at 36.8% fluorescence intensity. The decay time was observed to be similar for luminol doped POPD doped in various media, Table 5 . Table 5 Fluorescence data of POPD and Luminol doped POPDs

Sample

λmax

Asample Integrated

(nm)

Area

Quantum

Decay

Yield (ɸ) τ(ns)

621

0.018

4.74 x 107 0.13

4.75

POPD-Lum-NaOH 618

0.013

4.82 x 107

0.16

4.74

POPD-Lum-H2O2

615

0.011

4.41 x 107

0.18

4.74

POPD-Lum-H2O

613

0.016

5.42 x 107

0.19

4.73

POPD

The confocal micrograph of POPD, Figure 6(a), in solid state revealed emission in the red region which was correlated with strong peak at 521 nm showing scattered tiny particles. The confocal micrograph of POPD-Lum-H2O, Figure 6(b), showed brighter red particles as compared to pure POPD while POPD-Lum-NaOH, Figure 6(c), also revealed intense red fused particles. POPDLum-H2O2, Figure 6(d), exhibited bright red particles which formed dense agglomerates. The


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intensity of the red emission in all the cases could be well matched with the high quantum yield shown by the doped polymers.

(a)

(b)


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(c)

(d)

Figure 6 Confocal micrographs of (a) POPD, (b) POPD-Lum-H2O, (c) POPD-Lum-NaOH, (d) POPD-Lum-H2O2 Dose-dependent evaluation of anti-promastigote activity and determination of IC50 The in-vitro leishmanicidal activity of POPD and luminol doped POPDS against promastigote L. donovani, are shown in Table 6, Figure 7 in comparison to Miltefosine Hydrate as antileishmanial reference drug. POPD-Lum-NaOH exhibited the highest toxicity for intracellular persisting L.donovani parasites with CC50 values of 636.35 ± 41.4. Leishmanial activities of other doped polymers were also found to show higher CC50 values as compared to pure luminol as well as the reference reagent. Possibly, elements of the mitochondrial respiratory chain were targets for these polymers.


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Table 6 In-vitro anti-leishmanial activity of POPD and Luminol doped POPDs

Compounds

Avg. IC50 ± SD Avg. CC50 ±SD (µg/ml)

(µg/ml)

Miltefosine Hydrate 17.2 ± 3.5

37.4 ± 7.5

POPD-Lum-NaOH

41.46 ± 3.76

636.35 ± 41.4

POPD-Lum-H2O2

47.3 ± 11.6

607.1 ± 15.95

POPD-Lum-H2O

38.8 ± 1.4

624.6 ± 28.25

Luminol-H2O2

447 ± 77

582.3 ± 4.35

Figure 7 In-vitro anti-leishmanial activity of Luminol doped POPD


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Figure 8 In-vitro cytotoxic activities of luminol doped POPD against human THP-1 cell line The cytotoxic effect of luminol doped POPD on promastigotes of L. donovani, against human THP-1 cell line are shown in Figure 8. The concentration of 1000 µg/ml of POPD showed maximum cytotoxic effect on promastigotes of L. donovani. Result clearly revealed that by increasing the concentration of luminol doped POPDs, the viability of promastigotes was found to decrease. Even at the maximum concentration of 1000 µg/ml, about 48% cells were still alive. The viability was found to be more than 50% for POPD-Lum-NaOH and POPD-Lum-H2O2 when the concentration was take as 500 µg/ml while at concentrations of 62.5 µg/ml and 31.25 µg/ml, the viability was observed to be more than 80%. Therefore it can be speculated that the doped polymers are non-toxic and can be safely used for bioimaging as well as other biomedical applications.


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Live cell imaging of Leishmania cells

Figure 9 Live cell imaging of Leishmaina L. donovani using luminol doped POPD as a biomarker Since POPD-Lum-NaOH showed good anti-leishmanial activity, it was chosen as a fluorescent biomarker for targeting leishmania cells (Video given in supporting information). The live cell image of POPD-Lum-NaOH, Figure 9, revealed that these could be used to label the parasite to perform biological imaging and analysis. The organelles of L. donovani were observed to intensively interact with the polymer (Video given in supporting information). The percentage of fluorescent parasite-containing organelles was noticed to fluoresce in both red and green lasers. POPD-Lum-NaOH was found to be localized in the lumen of the organelles (Video given in supporting information).


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CONCLUSION POPD and luminol doped POPDs were successfully synthesized using luminol in acidic ,basic and neutral media. The modification of POPD was confirmed by UV analysis which revealed a change in the intensity of polaronic transition peak of POPD.POPD and dye modified POPDs were found to exhibit fluorescence in the NIR region and the intensity of emission was governed by the type of luminol used. Confocal microscopy of luminol doped POPDs revealed intense emission in the red region. The doped polymers were also evaluated for their activity against leshmania. Cytotoxicity studies revealed that the polymers were potentially non-toxic at lower dosages while live cell imaging of POPD-Lum-NaOH stained leshmania gave intense emission in red region. Luminol doped POPDs could therefore be used as a fluorescent biomarker to label the parasite to perform biological imaging and analysis.

ASSOCIATED CONTENT Supporting Information Density functional theory (DFT) calculations of luminol in neutral, acidic ,basic media using B3LYP/6-31G method; optimized geometries; charged distribution; molecular orbitals; oscillator strength; theoretical UV spectra; XRD data of POPD and luminol doped POPD; videos of live cell imaging of leishmania using POPD-Lum-NaOH biomarker.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Tel: +91-9810776242 ORCID ID: 0000-0001-7485-4103


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Notes The authors declare no competing financial interest. Acknowledgement The corresponding author Dr.Ufana Riaz also wishes to acknowledge the DST-SERB for granting major research project vide sanction number SB/S1-PC-070/2013. The coauthor Mrs. Sapana Jadoun is thankful to DST-SERB for granting Senior Research Fellowship under the said project. The authors also acknowledge the SAIF Facility at All India Institute of Medical Sciences (AIIMS), New Delhi, India for TEM analysis and also the Advance Instrumentation Research Facility at JNU for the confocal studies and the fluorescence time resolved analysis. References (1) Jaiswal, J.K.; Goldman, E.R.; Mattoussi, H.; Simon, S.M. Use of Quantum Dots for Live Cell Imaging. Nat. Methods, 2004, 1, 73 -78. (2) Xu, M.; Gao, Z.; Wei, Q.; Chen, G.; Tang D. Label-Free Hairpin DNA-Scaffolded Silver Nanoclusters for Fluorescent Detection of Hg2+ using Exonuclease III-assisted Target Recycling Amplification. Biosens. Bioelectron.2016, 79 (15), 411-415. (3) Chen, M-L.; Liu, J-W.; Hu, B.; Chen, M-L.; Wang, J-H. Conjugation of Quantum Dots with Graphene for Fluorescence Imaging of Live Cells. Analyst, 2011, 136(20), 42774283. (4) Lin,Z.; Lv,S.; Zhang,K.; Tang,D. Optical Transformation of a CdTe Quantum Dotbased Paper Sensor for a Visual Fluorescence Immunoassay Induced by Dissolved Silver Ions. J. Mater. Chem. B, 2017,5, 826-833.


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Table of Contents (TOC) graphic


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Influence of Luminol Doping of Poly(o-phenylenediamine) on the

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