Fabrication and photophysical study of photofunctional nanoporous alumina membrane (PNAM) were performed, and its application of photodynamic antimicrobial chemotherapy (PACT) was investigated. Nanoporous alumina membrane (NAM) was fabricated by two-step aluminium anodic oxidation process. Surface of the fabricated NAM was modified with organo-silane agent to induce covalent bonding between NAM and a photosensitizer (PtCP: [5,10,15-triphenyl-20-(4-methoxycarbonylphenyl)-porphyrin] platinum). PtCP was covalently bonded to the surface of the modified NAM by nucleophilic acyl substitution reaction process. The morphology and the photophysical properties of the fabricated PNAM were confirmed with field emission scanning electron microscope (FE-SEM), steady-state spectroscopies, and nanosecond laser-induced time-resolved spectroscopy. For the efficacy study of PNAM in PACT, an enveloped animal virus, vesicular stomatitis virus (VSV), was utilized as a target organism. Antiviral effect of the PNAM-PACT was measured by the extent of suppression of plaque-forming units (PFU) after the light irradiation. In the cultures inoculated with PACT-treated VSV, the suppression of PFU was prominent, which demonstrates that PNAM is a potential bio clean-up tool.
Reactive oxygen species (ROS) have been a subject of the extensive studies due to their broad applications such as photodynamic therapy (PDT), photovoltaic device, light harvest coating, and photocatalyst [
In this work, we report a fabrication of the photofunctional nanoporous alumina membrane (PNAM) and its photophysical property. The nanoporous alumina membranes were fabricated by two-step aluminum anodic oxidation process [
The fabrication procedure of the PNAM is shown in Scheme
Preparation of photofunctional nanoporous alumina membrane.
Surface morphologies of NAMs were observed by a field emission scanning electron microscopy (FESEM, JEOL, 6500F). Surfaces of the modified NAM and the fabricated PNAM were confirmed by ATR-IR spectra with a ATR-IR spectrophotometer (Nicolet, impact 400). Steady-state absorption and luminescence spectra were obtained by using a UV-Vis spectrophotometer (Hitachi, U-2800) and a spectrofluorimeter (Hitachi, F-4500), respectively. For the membrane sample, the diffuse reflectance spectra were recorded by a UV-Vis spectrophotometer (Jasco, V-550) equipped with an integrating sphere (Jasco, ISV-469). The Nd-YAG-pumped OPO laser (BMI, OP-901, 10 Hz, 5 ns FWHM pulse) was utilized as an excitation source for the detection of time-resolved singlet oxygen phosphorescence. The phosphorescence signal was collected at the perpendicular angle to the excitation beam and detected with a monochromator (Optometrics LLC, mini-chrom04) and NIR-PMT (Hamamatsu, H9170-45). The signal was acquired by 500 MHz digital oscilloscope and transferred to a computer for further analysis [
The established cell lines of A549, Vero, and vesicular stomatitis virus (VSV, Indiana serotype) were originally purchased from ATCC (Rockford, Md, USA) and have been maintained at −70°C and 1 atm. For the evaluation of the efficacy with PNAM-mediated PACT on viral inactivation, A549 cells were used throughout the experiments. For viral preparation, VSV was propagated in Vero cells. Both the cell lines were maintained in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (both supplied by Gibco RBL, Grand Island, NY, USA), penicillin (100 units/mL), and streptomycin (100
For a plaque-forming assay, A549 cells were seeded (2 × 105) in 6-well tissue culture plates in the growth media and allowed to form a confluent monolayer. All viruses that were treated with light only, PNAM only, and the irradiated PNAM in 1 mL of the solutions were inoculated into the cells and allowed to attach to the cells by rocking randomly for 1 h at 37°C under 5% CO2 humidified atmosphere. For a negative control, the cells treated with a phosphate-buffered saline (PBS) were used to assure the viability of the cells. The culture fluid was removed by aspiration and washed twice with MEM. Each well was overlaid with 2 mL of a mixture of 2% agarose and 2 × MEM (1 : 1) and then incubated for 48 h at 37°C under 5% CO2-humidified atmosphere. Thereafter, the cells were rinsed by PBS and fixed in 2% paraformaldehyde for 15 min. These were then rinsed twice with PBS for staining with crystal violet. The titers of virus were estimated by use of the plaque-forming assay [
SEM images of the fabricated pure NAM are shown in Figure
FE-SEM images of (a) the top surface view of nanoporous alumina membrane with the pore diameter of 250 nm and (b) its cross-section view.
The surface modification of NAM with hydrophobic silane, APTES, and PtCP are confirmed with ATR-IR spectra. Figure
ATR-IR spectra of (a) pure NAM, pure APTES, and the surface modified NAM with APTES, (b) pure PtCP, pure PNAM, and the surface modified NAM with APTES.
Diffuse reflectance UV-Vis absorption spectrum of PNAM is presented in Figure
(a) Absorption and (b) phorescence spectra of the pure PtCP in toluene solution (thin solid line) and PNAM (thick solid line). The absorption spectrum of PNAM is obtained by applying the Kubelka-Munk function to the diffuse reflectance spectrum. The excitation wavelength was 510 nm for the emission spectra.
The most critical factor for the proof of the singlet oxygen generation from PNAM is the direct detection of the phosphorescence from the singlet oxygen molecules generated by the photoexcited PNAM. The singlet oxygen phosphorescences of PtCP and the PtCP-bounded PNAM were measured at the detection wavelength of 1270 nm in air-saturated toluene and distilled aqueous solution. The measured phosphorescence signals are presented in Figure
Phosphorescence decays of the singlet oxygens from (a) the pure PtCP in toluene, (b) PNAM with pore diameters of 250 nm in toluene, and (c) PNAM with pore diameters of 250 nm in aqueous solution at the detection wavelength of 1270 nm. The solid lines are the fitted lines with a single exponential decay.
Vesicular stomatitis virus is a very well-characterized lipid-enveloped virus which, together with its sensitivity for photodynamic treatment, makes it a suitable candidate for the study of photodynamic inactivation [
Plaques formed in A549 cells by the VSV treated with PNAM and light (5 mW). Extent of the VSV-induced PFU in A549: (control) normal level of PFU in A549 cells caused by VSV, (light only) virus treated with light only without PNAM, (PNAM only) virus treated with PNAM only without light, and (PNAM w/Light) virus treated with PNAM and light
It has successfully been demonstrated that the singlet oxygen of highly oxidative species is generated from PNAM which is fabricated by the surface modification of NAM with organo-silane agents, APTES, and the photosensitizer of PtCP. The singlet oxygen lifetimes depending on the pore diameter of PNAM and the solvent environment were also investigated to understand their dynamics in restricted condition that directly affects the efficiency of PACT. The photo-irradiated PNAM for the application of PACT was performed with the inactivation of VSV. The VSV was significantly inactivated by PNAM-PACT under 5 mW of light condition. Therefore, it suggests that the developed PNAM from this study has a high possibility to be utilized as a photodynamic antiviral system. Furthermore, based on the experimental results of the controlled singlet oxygen lifetimes by the pore diameter of the membrane, this ROS generating nanoporous membrane can be developed as the spatial distance selective photocatalytic membrane for biological and environmental hazards as well as selective photocatalytic reaction.
This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Family Affairs, Republic of Korea. (grant no. A085136). The authors are grateful to Prof. H. K. Kim for the generous support of PtCP and Dr. S. I. Oh for the help in the preparation of NAM.