Photobiological Sciences
PAPER
Cite this:Photochem. Photobiol. Sci., 2019, 18, 1147
Received 19th October 2018, Accepted 8th February 2019 DOI: 10.1039/c8pp00470f rsc.li/pps
Water-dispersible glycosylated poly
(2,5
’-thienylene)porphyrin-based nanoparticles
for antibacterial photodynamic therapy
†
Rehan Khan,
a,bMelis Özkan,
‡
a,bAisan Khaligh
‡
a,band Dönüs Tuncel
*
a,bHere we report the preparation of water-dispersible glycosylated poly(2,5’-thienylene)porphyrin based nanoparticles by a nanoprecipitation method and demonstrate the application of these nanoparticles in antibacterial photodynamic therapy. The diameter of the nanoparticles is in the range of 50–80 nm and the resulting nanoparticles are stable in water without precipitation at least for a month. They have high singlet oxygen efficiency and display light-triggered biocidal activity against both Gram negative bacteria (Escherichia coli, E. coli) and Gram positive bacteria (Bacillus subtilis, B. subtilis). Upon white light irradiation for 10 min with a flux of 22 mW cm−2 of the E. coli suspension incubated with NPs (18 µg mL−1), a killing efficiency of 99% is achieved, whereas in the dark the effect is recorded as only around 8%.
Introduction
The development of new antimicrobial drugs and efficient therapeutic agents for the treatment of pathogenic infections is urgently needed because the antibiotic resistance of micro-organisms has become a serious global health issue.1,2 Light-triggered antibacterial treatment, the so called antimicrobial photodynamic therapy (APDT) or photodynamic antimicrobial chemotherapy (PACT), can be a very efficient way for killing microbial cells with resistance to antibiotics.3–5 In this method, the light, photosensitizer, and molecular oxygen are the main components to generate reactive oxygen species (ROS) including singlet oxygen that are mainly responsible for the antimicrobial activities and prevent the bacteria to develop further resistance.6,7Efficient photosensitizers that will gene-rate singlet oxygen in high yields for PACT are highly sought after. Porphyrin derivatives8–11 and recently conjugated oligo/
polyelectrolytes12–16 and nanoparticles17,18 have been found quite suitable for this purpose.
Among them, especially porphyrins are highly appealing as a photosensitizer because of their absorption in the visible range of the electromagnetic spectrum, long-living triplet excited state, high molar extinction coefficient and their ability to generate singlet oxygen when they are irradiated with light.8–11 An ideal photosensitizer should not have dark tox-icity and be water soluble.19However, many porphyrin deriva-tives are insoluble in water. Attaching hydrophilic groups such as amine, carboxylic acid, sulfonic acid and glycosyl units to the meso-positions of porphyrin is a commonly used approach,11,20–23but still the problem may persist as the por-phyrins have a large hydrophobic core which causes aggregate formation through π–π interactions and the hydrophobic effect. This, in turn, decreases their singlet oxygen efficiency.
Another strategy is to convert directly the hydrophobic por-phyrin derivatives into water dispersible nanostructures.24–34 This approach is especially highly attractive because photosen-sitizers which are ionic and carry charges usually exhibit dark toxicity and are effective on Gram positive bacteria but not on Gram negative bacteria.35A number of porphyrin-based nano-structures have been reported, however, they usually have a limited dye-loading content and as a result, low singlet-oxygen production efficiency as a photosensitizer. Therefore, exploring alternative strategies for the design of porphyrins with good biocompatibility and excellent water dispersibility is highly desirable.
In this context, here we report water-dispersible and stable nanoparticles prepared through glycosylated
poly(2,5′-thieny-†Electronic supplementary information (ESI) available: Full synthetic scheme for PTTP-Glu-Ac, synthetic procedure for PTTP,1H,13C-NMR spectra of PTTP, 1H, 13C-NMR, FT-IR, UV-Vis, PL spectra of PTTP-Glu-Ac, time-dependent decrease of absorbance spectra for DPBF with NPs, minimum inhibitory concen-tration plots of NPs against E. coli in the dark and under light, plate photographs for NPs against B. subtilis on YTD agar plate in the dark and under light. See DOI: 10.1039/c8pp00470f
‡These authors contributed equally.
aDepartment of Chemistry, Bilkent University, 06800 Ankara, Turkey. E-mail: dtuncel@fen.bilkent.edu.tr
bInstitute of Materials Science and Nanotechnology, National Nanotechnology Research Center (UNAM), Bilkent University, Ankara, 06800, Turkey
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lene)porphyrin based nanoparticles for antibacterial photo-dynamic therapy. Glycosylation is expected to provide stability as well as additional benefits such as cell–surface interactions and receptor targeted cargo delivery.11,23,36–38 The acetyl groups are deliberately kept intact to be able to control the sizes of nanoparticles, further enhance their stability and improve cell permeability through the hydrophobic effect39 and after entering the cells they are known to be removed by nonspecific intracellular esterases. These nanoparticles show high singlet oxygen efficiency and do not show toxicity in the dark but they become phototoxic when they are irradiated with light. They show a light-triggered antibacterial effect even at a low concentration and exposure to light for short time.
Experimental section
Materials
All chemicals used in the syntheses were of analytical grade, obtained from Sigma-Aldrich, and used as received. Milli-Q water (18.2 MΩ cm at 25 °C) was used when needed. Solvents were dried and distilled before use. All reactions were per-formed under air unless otherwise stated. Thin layer chromato-graphy was performed on SiO2 60 F-254 plates and flash
column chromatography was carried out using SiO2 60 (
par-ticle size 0.040–0.055 mm, 230–400 mesh). NMR spectra (1H, at 400 MHz and13C at 100 MHz) were recorded on a Bruker DPX-400 spectrometer in CDCl3 and DMSO-d6 solvent and
TMS (δ = 0.00 ppm) as an internal standard. Chemical shifts were reported asδ values in ppm as referenced to TMS. FT-IR spectra were recorded with a Bruker Alpha-II Platinum ATR FT-IR spectrometer. The mass spectra were recorded using Agilent 6224 High Resolution Mass Time-of-Flight (TOF) LC/ MS by the electrospray ionization method. UV-VIS absorption spectra were recorded on a UV–vis spectrophotometer (Cary UV–vis) with 1 cm path length quartz cuvettes in the spectral range of 300–800 nm. Emission spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse Fluorescent spectrophotometer). The quantum yields of fluorescence of the compounds were determined using the integrated sphere method. Morphologies of polymer nanoparticles were investi-gated using focused ion beam scanning electron microscopy (FEI, NanoSEM) and transmission electron microscopy (FEI Technai G2 F30). TEM images were acquired with a TEM, FEI Tecnai G2 F30. TEM grids were prepared by placing 1μL of the particle solution on a carbon-coated copper grid and drying at room temperature. Determination of the size distribution and average diameter of nanoparticles with respect to their hydro-dynamic sizes was performed via hydro-dynamic light scattering (DLS) measurements (Malvern Nano-ZS Zetasizer).
Synthesis of poly-5,15-diphenyl(2,5 ′-dithienylene)-10,20-di(3,5-di-O-TEG-phenyl)porphyrin (PTTP)40
In a 50 mL two-neck round bottom flask Porphyrin 239 (350 mg, 0.245 mmol) and 5,5 ′-bis(tributylstannyl)-2,2′-bithio-phene (180 mg, 0.245 mmol) were dissolved in anhydrous
toluene : DMF mixture (2 : 1, v/v, 10 mL) and degassed through three freeze–pump–thaw cycles. After stirring for 15 min, cata-lyst Pd(PPh3)4(12.2 mmol) was added and the resulting
reac-tion mixture was refluxed under argon at 90 °C for 48 h. After the reaction was over, the mixture was cooled down and preci-pitated in cold MeOH. The precipitates were collected by fil-tration and washed with MeOH (3–4 times) followed by n-hexane. The precipitates were redissolved in chloroform and precipitated in cold methanol. The polymer was obtained as a purple solid (57% yield).1H-NMR (400 MHz, CDCl3, 25 °C):δ
9.05–8.95 (m, pyrrolic–H), 7.75–8.02 (m, Ar–H), 7.56–6.85 (m, Ph–H), 3.98 (broad peak, TEG–CH2). 13C-NMR (100 MHz,
CDCl3, 25 °C):δ 158.67, 150.03, 144.03, 135.77, 135.03, 132.13,
131.66, 130.90, 129.71, 128.83, 125.60, 124.78, 124.59, 124.23, 123.64, 113.86, 69.37, 55.55, 53.55. Mn= 3109, Mw/Mn = 1.21;
UV-vis (CHCl3):λmax(nm); 431, 557, 604.
Synthesis of poly-5,15-diphenyl(2,5 ′-dithienylene)-10,20-di(3,5-di-O-TEG-tetra-O-acetyl-D-galactopyranoside-phenyl) porphyrin
(PTTP-Glu-Ac)
β-D-Glucose pentaacetate (1.17 mmol, 452 mg) and poly(2,5
′-thienylene)porphyrin (PTTP) (0.146 mmol, 200 mg) were dis-solved in 20 mL anhydrous dichloromethane in a two neck round-bottom flask (r.b.). BF3·OEt2 (0.87 mmol, 100 µL) was
slowly added to r.b. under N2. The reaction mixture was stirred
at room temperature for 4-days and then poured into a satu-rated aqueous solution of NaHCO3(25 mL). The organic phase
was separated and further extracted with water and brine. The combined organic phase was washed twice with water and fil-tered through a small plug of silica and evaporated to dryness. The solid residue was dissolved in THF (10 mL) and the result-ing solution was precipitated into a methanol–water mixture (1 : 2, v/v, 150 mL). Precipitates were collected by filtration and dried in vacuo. The product was obtained as a purple solid (210 mg, 70% yield).1H-NMR (CDCl3, 400 MHz, 25 °C)δ: 9.76
(s, pyrrolic–H), 8.96–8.06 and 7.42–6.91 (m, Ph–H), 7.65–7.52 (m, thiophene–H), 5.48–5.51 (m, glucosyl–CH), 5.40–3.65 (m, PEG–CH2and glucosyl–CH), 1.94–2.11 (s, –CH3),−2.80 (s,
pyr-rolic–NH). 13C-NMR (100 MHz, CDCl3, 25 °C): δ: 170.22, 158.94, 144.02, 143.95, 143.01, 141.57, 141.50, 139.81, 135.22 133.52, 131.23, 129.95, 129.00, 125.69, 124.98 124.53, 123.91, 122.50, 119.98, 119.66, 113.95, 100.23, 89.11, 69.88, 69.24, 67.96 61.75, 55.65, 29.70 UV-vis (DMF): λmax(nm); 423, 517, 555, 592, 648.
Preparation of PTTP-Glu-Ac nanoparticles
Nanoparticles were prepared by the reprecipitation method. First PTTP-Glu-Ac (10 mg) was dissolved in THF (10 mL) to prepare a stock solution with a concentration of 1 mg mL−1. In a typical nanoparticle preparation, the stock solution was injected into 100 mL of de-ionized water under sonication and the mixture was stirred in an ultrasonic bath further for 30 min. After the nanoparticle preparation THF was removed from the dispersion and the final volume of the nanoparticle dispersion was decreased to 10 mL under reduced pressure.
Batch 1: 5 mL stock solution was used and the final concen-tration was 0.5 mg mL−1; Batch 2: 3 mL stock solution was diluted with 2 mL THF and the final concentration was 0.3 mg mL−1; Batch 3: 2 mL stock solution was diluted with 3 mL THF and the resulting solution and the final concentration is 0.2 mg mL−1; Batch 4: 1 mL stock solution was diluted with 4 mL THF and the final concentration was 0.1 mg mL−1.
The Z-Average size, PDI and zeta potential of PTTP-Glu-Ac nanoparticles with different concentrations of PTTP-Glu-Ac solution are compiled in Table 1.
Stabilities of the nanoparticle dispersions were tested over time and the size and surface charges were recorded using DLS measurements.
Measurement of singlet oxygen production efficiency
Solutions containing 1,3-diphenylisobenzofuran (DPBF) (20μM) and PTTP-Glu-Ac NPs (0.5 μM, per repeating unit) or 5,10,15,20-tetrakis(1-methyl-pyridinium-4-yl)porphyrin (TPPy) (0.5 μM) in D2O : DMF (15 : 1) were mixed in the dark in a
quartz cuvette cell of 1 cm at room temperature with gentle stirring and then, the solutions were irradiated at 430 nm with a monochromator integrated Xenon lamp at pre-established irradiation intervals. Singlet oxygen generation was followed by time-dependent decrease of the absorbance at 418 nm due to the oxidation of DPBF. In order to take into account the poss-ible decomposition of DPBF in water, we carried out a control experiment in which DPBF solution in DMF was added to D2O
to obtain a solution with the final ratio of D2O : DMF (15 : 1)
and the resulting solution was irradiated at 430 nm with a monochromator integrated Xenon lamp at pre-established irradiation intervals used for the one with PTTP-Glu-Ac NPs and no significant decomposition of DPBF (and decrease in the absorbance band) was observed under these conditions. The relative ΦΔ 1O
2 generation efficiency was determined in
comparison with 5,10,15,20-tetrakis(1-methyl-pyridinium-4-yl) porphyrin (TPPy) by monitoring the reduced loss of absor-bance of DPBF (at 418 nm in water) with increasing irradiation time. The relationship between DPBF’s absorption value ratio (A/A0) and irradiation time indirectly reflected the1O2yield of
TPPy compared with PTTP-Glu-Ac NPs. The following eqn (1) was used to calculate the singlet oxygen quantum yield of PTTP-Glu-Ac NPs:
ΦNPs¼ ΦStðmNPs=mStÞðFSt=FNPsÞ ð1Þ
where the subscripts‘NPs’ and ‘Std’ denote PTTP-Glu-Ac NPs and TPPy, respectively;Φ (1O2) is the singlet oxygen quantum
yield, m is the slope of a plot of difference in change in absor-bance of DPBF (at 418 nm) with the irradiation time (Fig. S8, ESI†) and F is the absorption correction factor, which is given by F = 1–10−OD(OD at the irradiation wavelength).41
Determination of the minimum inhibitory concentration (MIC)42
The respective minimum inhibitory concentration (MIC) of nanoparticles was determined by the broth microdilution method. Hereby, a single colony of Escherichia coli(E. coli) on a solid Luria–Bertani (LB) agar plate was transferred to 5.0 mL of a liquid LB culture medium and was grown at 37 °C for 16 hours. The bacterial mixture was diluted 2-fold with pure LB and the initial OD value was adjusted to 1.1 and 6 different concentrations (from 0.2 mg mL−1nanoparticle stock solution in distilled deionized water) were prepared as follows: 4.6, 9.1, 13.7, 18.2, 22.8 and 27.3 µg mL−1. 1.5 mL-Eppendorf tubes were inoculated with 100 µL of the bacterial mixture and then 10 µL from each concentration was added. To carry out the positive control, equal volume of distilled deionized water was used. Both blank and nanoparticle containing tubes were exposed to white light for 10 minutes with a flux of 22 mW cm−2. The same experiment was done in the dark as well. After light and dark exposure, Eppendorf tubes were placed in an incubator at 37 °C with shaking (200 rpm) for 16 hours. At the end of the incubation period, the bacterial mixtures were placed from Eppendorf tubes to a 96-well plate. With the microplate reader, the optical density at 600 nm was measured. The results were repeated in triplicate.
Investigation of antibacterial activities of nanoparticles toward Escherichia coli (E. coli)
A single colony of E. coli on a solid Luria–Bertani (LB) agar plate was transferred to 5.0 mL of the liquid LB culture medium and was grown at 37 °C for 16 hours. Bacteria were harvested by centrifuging (7000 rpm for 2 min at 4 °C) and washing with PBS three times. The supernatant was discarded and remaining E. coli were resuspended in PBS. The optical density at 600 nm (OD600) of the bacterial suspension was
adjusted to 1.0. Then the suspension was diluted 5-fold with PBS. 200 μL nanoparticle dispersion (concentration of stock nanoparticle dispersion is 0.2 mg mL−1) was mixed with 2 mL E. coli suspension (concentration of nanoparticles is now 18 µg mL−1) and the mixture was incubated for 15 minutes in the dark at 37 °C and then irradiated with white light with a flux of 22 mW cm−2for 10 minutes. Then the bacterial suspen-sion was serially diluted (104fold) in PBS. A 50 μL portion of the diluted bacterial E. coli was spread on the solid LB agar plate. The colonies formed after 16 hours of incubation at 37 °C were quantified. The same procedure was repeated for the nanoparticles incubated with bacteria in the dark without exposure to light. Control experiments were carried out with exposure to light and in the dark in the absence of nanoparticles.
Table 1 Z-Average size, PDI and zeta potential of PTTP-Glu-Ac nano-particles with different concentrations of PTTP-Glu-Ac solution Batch Conc. (mg mL−1) Z-Average (d nm) PDI Z-Potential (mV) 1 0.5 79.1 ± 4.1 0.32 −32.3
3 0.3 75.4 ± 9.2 0.23 −36.4 4 0.2 58.4 ± 0.9 0.21 −37.8 5 0.1 56.2 ± 1.1 0.21 −38.0
Antibacterial activities of nanoparticles toward Gram posi-tive bacteria (Bacillus subtilis, B. subtilis) were also studied fol-lowing the similar procedure described for E. coli.
Results and discussion
First, poly-5,15-diphenyl(2,5 ′-dithienylene)-10,20-di(3,5-di-O-TEG-phenyl) porphyrin (PTTP) was synthesized as shown Scheme 1 and characterized by1H and13C NMR spectroscopy (Fig. S1 and S2†). The number average (Mn) and weight average
(Mw) molecular weights of the polymer were determined by
GPC as 3109 and 3549 Da respectively. The values are prob-ably lower than the real values because of the difficulty of molecular weight determination of rigid polymers like this one. ESI-MS spectrometry also showed doubly charged peaks around 3549 Da (Fig. S3†). Then, PTTP was glucosylated using excess β-D-glucose pentaacetate in the presence of a
Lewis acid (BF3: Et2O) as shown in Scheme 1. Glucosylated
polymer, PTTP-Glu-Ac was isolated as a purple solid first
eluting its dichloromethane solution over a pad of silica and then precipitating its concentrated solution into a non-solvent.
1H-NMR, 13C-NMR and FT-IR spectra of the isolated solid
prove the formation of PTTP-Glu-Ac (Fig. S5–S7, ESI†). FT-IR shows the characteristic band at 1740 cm−1 for carbonyl groups on the acetyls of glucosyl units (Fig. S5, ESI†). The pres-ence of a chemical shift at 170 ppm due to the carbonyl carbon of the acetyl groups in the 13C-NMR spectrum also further confirms the success of the glucosylation reaction (Fig. S7, ESI†). The1H-NMR spectrum clearly reveals the peaks
in the region of 4.3–5.5 ppm due to glucosyl protons and especially the characteristic peaks at around 1.8–2.3 ppm due to acetyl (CH3) protons are evident (Fig. 1). From the
inte-grations we can estimate the number of attached glucosyl units to porphyrin for each repeating unit approx. as 3. It should also be noted that during glycosylation demetallation (removal of Zn) took place due to the acidic environment and the chemical shift at−2.8 ppm for the pyrrolic proton (–NH) confirms this (Fig. S6 and S1†).
Scheme 1 Synthesis schemes for poly-5,15-diphenyl(2,5’-dithienylene)-10,20-di(3,5-di-O-TEG-phenyl) porphyrin (PTTP)39and PTTP-Glu-Ac by the glucosylation reaction of PTTP withβ-D-glucose pentaacetate.
We prepared nanoparticles from the glucosylated polymer PTTP-Glu-Ac through the reprecipitation method. In this method, glucosylated porphyrin was first dissolved in THF at predetermined concentrations and the resulting solutions were injected into water under ultrasonication. The mixture was further sonicated about 30 min. Thereafter, THF was removed under reduced pressure to obtain water dispersible glycosylated porphyrin polymer nanoparticles. The size of the nanoparticles can be controlled by tuning the reaction con-ditions (e.g. concentration of polymer and the volume of the poor solvent, etc.). The volume of the aqueous dispersion of nanoparticles can be further decreased to obtain the desired
concentrations under reduced pressure without any changes on the initial sizes of the nanoparticles. Dynamic light scatter-ing (DLS) was used for the initial characterization of the nano-particles. The nanoparticles we prepared fall within the optimal range of 50–80 nm as shown in Table 1.
For the further investigation we selected the nanoparticles prepared in batch 3. Fig. 2a shows the DLS measurement of the nanoparticles with an average size of 58 nm and a narrow size distribution (PDI) of 0.21 in water. The surface charge of the prepared nanoparticles in water was also measured as a zeta potential value by DLS as −38 mV (Fig. 2b). This value indicates the high stability of the nanoparticle dispersion. The concentration of these nanoparticles is 0.2 mg mL−1. At higher concentration the sizes of the nanoparticles increase but their stability decreases (Table 1). Sizes and the shapes of these nanoparticles were further investigated by using electron microscopes (SEM and TEM). As can be seen from the TEM images of nanoparticles (Fig. 2c), nanoparticles have a spheri-cal shape and their sizes are around 50–60 nm. In order to investigate the stability of nanoparticles in water, their sizes were recorded over one month using DLS measurements. No significant changes were observed in their sizes. They appeared to be very stable and remain clear without precipi-tation at least for a month.
Fig. 3 shows UV-vis absorption spectral changes of PTTP-Glu-Ac solution in DMF and nanoparticle dispersion in water. PTTP-Glu-Ac solution in DMF exhibited a sharp Soret band at 423 nm and four weak Q-bands at 517, 555, 592, and 648 nm as typical absorption peaks of free base porphyrins. The absorption bands of the nanoparticles in water 4–5 nm
Fig. 1 1H-NMR spectrum (400 MHz, CDCl
3, 25 °C) of the post-functio-nalized porphyrin–polymer.
Fig. 2 (a) DLS histogram showing the particle size distribution by intensity, (b) zeta potential measurements by DLS, (c) TEM image of the nano-particles (the scale bar: 50 nm and for inset image 200 nm).
red-shifted and the Soret band became slightly broader than that of PTTP-Glu-Ac solution in DMF. It meansπ–π interaction is limited due to the presence of bulky substituents and thi-enylene groups connected to porphyrin cores and the
por-phyrin-based chromophore is able to preserve its photo-physical properties.28,29The excitation of the nanoparticles in water at 427 nm resulted in fluorescence emission above 600 nm as is characteristic of porphyrin with two vibrational bands at 658 and 723 nm (Fig. 3). Fluorescence quantum yield of the polymer, PTTP-Glu-Ac, solution in DMF was measured as 7% but upon converting the polymer into nanoparticles the yield was decreased to around 1%. The low fluorescence quantum yield of nanoparticles in water can be explained by both inter- and intrachain interactions upon collapsing polymer chains into nanoparticles, as well as the solvent polarity.
Singlet oxygen production efficiency of the nanoparticles was determined using 1,3-diphenylisobenzofuran (DPBF) as an efficient 1O2 trap in combination with accurate,
time-depen-dent spectrophotometric determination of the DPBF concen-tration. The relative ΦΔ 1O2 generation efficiency was
deter-mined in comparison with 5,10,15,20-tetrakis(1-methyl-pyridi-nium-4-yl)porphyrin (TPPy) by monitoring the reduced loss of absorbance of DPBF (at 418 nm in water) with increasing irradiation time (Fig. S8, ESI†). The relative magnitude of singlet oxygen generation efficiency was examined by means of TPPy as a reference. In the literature the1O2quantum yield of
TPPy was reported as 0.58 in water.43,44The1O2quantum yield
Fig. 3 UV-vis absorption and fluorescence spectra of PTTP-Glu-Ac solution in DMF and nanoparticles in water excited at 427 nm.
Fig. 4 (a) Linearized plots based on the decrease in the absorbance intensity of DPBF in the presence of NPs and TPPy irradiated at 430 nm with 15 s intervals; (b) biocidal activities of NPs towardE. coli in the dark and under photo-irradiation. The values represent the mean ± standard deviation (SD) of six separate experiments. Error bars represent SD of data from six separate measurements. Plate photographs forE. coli on a YTD agar plate treated (c) without the photosensitizer in the dark, (d) with the photosensitizer in the dark, (e) without the photosensitizer under photo-irradiation (with aflux of 22 mW cm−2white light for 10 min), (f ) with the photosensitizer under photo-irradiation (with a white lightflux of 22 mW cm−2for 10 min).
of the nanoparticles is calculated to be around 85%, which is significantly higher than that of TPPy.
After finding out that NPs have the ability to generate singlet oxygen in high yields, we expected them to show also efficient light-triggered biocidal activities. For this purpose Escherichia coli (E. coli), a Gram-negative bacteria, which is responsible for half of the infections was selected as a model and its interactions with nanoparticles were investigated. Bacterial survival experiments were carried out using various concentrations of NPs in the dark and upon exposure to white light for 10 min with a flux of 22 mW cm−2 by a surface plating method18(Fig. 4c–f). Colony counting showed that the killing efficiency upon irradiation of the E. coli suspension incubated with NPs (18 µg mL−1) was 99%, whereas the killing efficiency in the dark is only around 8% (Fig. 4b). Studies of the effect of NP concentrations on the killing efficiency toward E. coli under irradiation showed that the killing efficiency enhanced with increasing concentration of NPs and reaches a plateau after 18 µg mL−1(Fig. S10 and S11, ESI†).
The light-triggered antibacterial effect of NPs against the Gram positive bacteria (B. subtilis) was also investigated under the same conditions (concentration of NPs, light intensity, and exposure time). The results show that the killing efficiencies of NPs on B. subtilis under light and in the dark are similar to the one observed on E. coli (Fig. S12†). These findings suggest that these nanoparticles can be used as a broad-spectrum antibac-terial agent.
Recently Wu and co-workers reported that the interaction of E. coli with NPs which show a negative zeta potential is not due to electrostatic interaction but rather through hydrophobic interaction.39 Our NPs were prepared from hydrophobic, neutral polymers and they are dispersible in water. Although they have a negative zeta potential, they are not ionic. The interaction of NPs with bacteria probably through the hydro-phobic effect as reported by Wu and co-workers and these NPs are effective both on Gram positive and Gram negative bac-teria. Moreover, the presence of sugar units of nanoparticles may facilitate cellular recognition via specific carbohydrate protein interactions on the bacteria surface.11 Consequently, when the nanoparticles are irradiated with light, the generated singlet oxygens diffuse more effectively and kill the bacteria.
Conclusion
In summary, we have synthesized a glucosylated porphyrin– thiophene based photoactive polymer and converted it into nanoparticles using the nanoprecipitation method. These nanoparticles are water-dispersible with particle sizes in the range of 50–80 nm and these were also found to be stable in water over months. They have ability to generate singlet oxygen in high yields and show light-triggered antibacterial activity against the Gram negative bacteria, E. coli as well as Gram positive bacteria, B. subtilis. They confer the ideal photosensiti-zer requirements as they do not have dark toxicity and are water dispersible.
In this regard, these nanoparticles are ideal for photo-dynamic antibacterial therapy and can be used as a broad-spectrum antibacterial agent. Moreover, it would be possible to load drugs to these nanoparticles in order to combine chemotherapy with photodynamic therapy.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Con
flicts of interest
There are no conflicts to declare.
Acknowledgements
We thank for the financial support to the Scientific and Technological Research Council of Turkey (TUBITAK) grant numbers 112T058, 215Z035.
Notes and references
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