Chemicals
Amberlite®IR120 strong acid cation exchange resin, 5-ALA and acetylacetone were purchased from the Sigma-Aldrich, USA. Hydrochloride acid was purchased from Fluka, Switzerland. Ammonium hydroxide was purchased from Thermo-Fisher, USA. Activated carbon was ordered from Alfa Aesar, USA. Phosphoric acid was purchased from Merck, USA. Acetone, diethyl ether, ethanol, and methanol were purchased from ECHO, Taiwan. 4-Dimethylaminobenaldehyde (DMAB) was ordered from ACROS Organics™. Perchloric acid and sodium acetate were purchased from SHOWA, Japan.
Culture condition of 5-ALA
Biofabrication of 5-ALA was carried out by culturing strain RcI from a previous publication (Yu et al. 2022). RcI was precultured in Luria–Bertani (LB) medium at 37 °C, 200 rpm for 16 h. The preculture cell was inoculated with 2% (v/v) into 300 mL MM9 medium containing (NH4)2SO4 (16 g/L), Na2HPO4•12H2O (16 g/L), KH2PO4 (3 g/L), yeast extract (2 g/L), MgSO4•7H2O (0.5 g/L), MnSO4•7H2O (0.01 g/L), glucose (20 g/L), and glycerol (10 g/L) in a 1-L bioreactor at 37 °C, 300 rpm with 1 vvm aeration. The final concentration of 0.1 mM IPTG, 0.4 mM ferric citrate, 4 g/L glycine, 1 g/L succinate and 30 μM PLP were added in cultivation when OD600 reached 0.6–0.8 and shifted the culture to 30 °C and 500 rpm until 24 h. Substrates including 3 g/L glycine, 1.5 g/L glucose, and 2 g/L succinate were fed at 12 h. The cell concentration was measured by a spectrometer (SpectraMax 340, Molecular Devices, USA) with an optical density at 600 nm (OD600).
Ehrlich assay for quantification of 5-ALA
A 200 μL 5-ALA sample was mixed with 200 μL sodium acetate (pH 4.6) and 40 μL acetylacetone, then the mixture was heated at 100 °C for 10 min to accelerate the reaction (Yu et al. 2022). After cooling to room temperature, the mixture was mixed and reacted with the same volume of Ehrlich’s reagent for 10 min in dark. Finally, the solution was analyzed by optical density at wavelength 553 nm by using a spectrophotometer.
Purification of 5-ALA using chromatography
The strong acid cation exchange resin (Amberlite® IR120) was packed in a column (7.07 cm2, 20 cm height). First, the resin was immersed in 50 mL of 1.5 M HCl for 1.5 h, followed by 50 mL of 1.5 M NaOH. A 50 mL of 1.5 M HCl was passed through the column to prepare an H+-form condition. The resin was washed by ddH2O once between each step. The culture broth of 5-ALA was adjusted to pH 4.2–4.8 with acetate acid before adsorption. A 600 mL broth was applied to the column and then 100 mL ddH2O passed through to wash out the residual medium. HCl, sodium acetate buffer (SAB) and ammonia were applied in this study to examine the efficiency of 5-ALA desorption with different concentrations and different pH. Finally, 85% phosphate acid was added into the desorbed 5-ALA solution and adjust the pH to 3.0.
Crystallization of 5-ALA
To remove the impurities in the broth, different amount of activated carbon was added into the solution and stirred at 500 rpm for 30 min for decolorization. The solution was then concentrated in a rotary evaporator to obtain a higher concentration of 5-ALA (i.e., 250 to 500 g/L), which was dripped into the different organic solvents including diethyl ether, methanol, ethanol, or acetone (Tachiya 2016). Finally, the precipitate was dried in the vacuum dryer (EYELA, Japan).
HPLC analysis
The high-performance liquid chromatography (HPLC, Hitachi, Japan) was employed to analyze the purity of 5-ALA precipitate. Derivatization of samples were performed by the reaction consisting of 680 μL of 0.05 M borate buffer (pH 9), 480 μL of 100% methanol, 12 μL sample and 30 μL of 200 mM diethyl ethoxymethylenemalonate (DEEMM). The samples were heated at 70 °C for 2 h to complete the degradation of excess DEEMM and derivatization. Afterward, the samples were placed into HPLC with a quaternary pump, an inline degasser, an autosampler, and a column thermostat. Chromatographic separation was carried out by reverse-phase chromatography on a C18 column (YMC-C18 column, 4.6 × 250 mm, 5 μm particle size), maintained at 35 °C. Mobile phase A was composed of 100% acetonitrile, and B was made up of 25 mM aqueous sodium acetate buffer (pH 4.8). The flow rate of 0.8 ml/min was used, with the following gradient program: 0–2 min, 20–25% A; 2–32 min, 25–60% A; 32–40 min, 60–20% A. Detection was carried out at 284 nm (Xue et al. 2020).
Cancer cell culture and photodynamic therapy
Human lung adenocarcinoma cells (A549 cells) and melanoma skin cancer cells (A375 cells) were purchased from Bioresource Collection and Research Center (BCRC, Taiwan), and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA). All cells were incubated in 10-cm tissue culture dishes at 37 °C and 5% (v/v) CO2. The cancer cells were seeded in 96-well plates using a fresh DMEM culture medium, then incubated under 37 °C and 5% (v/v) CO2 for 24 h before being treated by 5-ALA-PDT. The cells were incubated for 3 h with different concentrations of 5-ALA (0, 5, 10 g/L). Thereafter, the cells were exposed to the red light source at 635 nm with power density 100 J/cm2 for 15 min (HUA YANG Precision Machinery Co., Taiwan). After PDT treatment, the cells were incubated at 37 °C and 5% (v/v) CO2 for different time (0, 2, 4, 12 h) to make reactive oxygen species attack cells. Finally, the CCK-8 assay was applied to identify the viability of the cells. Before performing the cell counting kit-8 (CCK-8) assay, the culture medium consisting of 5-ALA was removed due to the background value. 10 µL of the CCK-8 reagent (MedChemExpress Ltd.) and 100 µL of the DMEM were added to each well, incubated the cells were at 37 °C and 5% (v/v) CO2 for 1 h, and optical density at 450 nm was measured using a spectrophotometer. Statistical analysis was performed using GraphPad Prism software version 8.0 (GraphPad Prism software, San Diego, USA). Differences in cell viability among the groups were analyzed using a t-test, and the values of p < 0.05 were statistically significant.
Antibacterial photodynamic therapy (aPDT) against pathogens
The elimination of P. hauseri by aPDT was carried out with minor modifications from a previous study (Yi et al. 2021b). The pathogen was incubated in a 10 mL LB medium at 37 °C and 175 rpm for 16 h. The concentration of P. hauseri was adjusted to OD600 at 0.2 (approximately 109 cells/mL) and injected 180 µL into 96-well plates. An appropriate amount of 5-ALA solution (i.e., 0.25%, 0.5% and 1%) was added to the cell sequentially. The plates were wrapped with aluminum foil to avoid the light and incubated at 37 °C for 3 h to metabolize 5-ALA to PPIX. Subsequently, it was illuminated with or without LED red light at a wavelength of 635 nm for 30 min, corresponding to power density of 100 J/cm2 for aPDT (HUA YANG Precision Machinery Co., Taiwan). Finally, to calculate the colony-forming unit (CFU), a 20-µL aliquot with a dilution rate of 10–1 to 10–5 was spread onto an agar plate and incubated at 37 °C for 16 h. The A. hydrophila, Bacillus cereus (B. cereus), Staphylococcus aureus (S. aureus) were tested by following the procedure as aforementioned. Each experiment was carried out in triplicate.
aPDT in the algal culture
Chlorella sorokiniana (Cs) was precultured in TAP medium for 2 days to reach OD680 at 0.8 (Lin et al. 2021). A. hydrophila was precultured in LB medium at 37 °C, 200 rpm for 16 h. The mixture of 5-ALA, A. hydrophila and Cs were prepared in a 10 mL TAP medium and cultured under white light (100 µmol/m2/s) with shaking at 150 rpm. The final600 concentration was adjusted to OD680=0.08 for Cs, and 0.2, 0.3, or 0.4 of OD600 for A. hydrophila, while the 5-ALA concentrations were used by 0.05%, 0.1% and 0.2%, respectively. Afterward, a 20-µL aliquot was dropped on the TAP plate for a 3-day cultivation to observe the growth of Cs and A. hydrophila.