Hypericin

Hydrophilic aluminium(III) phthalocyanine chloride tetrasulphonate (AlPcS₄Cl), molecular weight 895.19 g/mol, (Frontier Scientific, Logan, UT, USA), and Hypericin, molecular weight 504.44 g/mol (Sigma-Aldrich, 56690-1 MG), were used. Stock solutions of 100 μM AlPcS₄Cl were solubilized in Dulbecco's phosphate buffered saline (DPBS; Sigma-Aldrich, D8537) and 2 mM Hypericin were solubilized in dimethyl sulfoxide (DMSO, BDM Merck, Germany) and sterilized using a 0.2 μm filter.

Hypericin, low molecular weight chitosan (stabilizer and viscosity enhancer) were purchased from Sigma Aldrich chemie Gmbh USA (St. Louis, MI, USA), eucalyptus oil (The major constituents of eucalyptus leaves essential oils are 1,8-cineol ranging from 49.07 to 83.59%, and α-pinene in the range of 1.27 to 26.35%), ethanol, Tween^® 80, Span^® 80, and distilled water were purchased from Dawaa Al-Gharbyah, KSA. All of the chemicals used were of the highest analytical grade.

Acetonitrile, methanol, ortho-phosphoric acid (HPLC grade), rutin, hyperoside, hypericin and hyperforin were purchased from Sigma-Aldrich, Germany for phytochemical profiling. Quercetin dihydrate (≥98%) was obtained from Carl Roth (Karlsruhe, Germany). Hydroxyethyl cellulose (HEC), hydroxy propylmethyl cellulose (HPMC), sodium carboxymethyl cellulose (NaCMC), and Pluronic F-127 were purchased from Sigma-Aldrich, Germany for the formulations. Ketamine HCl 5% (Rotex medica, Trittau, Germany), and Panthenol® 2% cream (Nile Co. for Pharmaceuticals and Chemical Industries, Egypt) were obtained for the in vivo experiments. Formaline, xylene, hematoxylin and eosin stain, and Masson’s trichrome stain were sourced from Sigma-Aldrich, Germany for the histopathology. All other chemicals and reagents were of analytical grade.

The hypericin used in this study was purchased from Sigma Aldrich, Johannesburg, South Africa (Catalogue: 1MG-56690). A working stock solution of 2 mM in 1 mL was prepared using dimethyl sulfoxide (DMSO) in the final solution, and stored at 4 °C, covered in foil for protection from direct light. AuNPs were purchased from Sigma Aldrich (Catalogue: 1ML-765457), the particles were 10 nm in size and functionalized with PEG 3000 containing a carboxylic acid terminal. The stock concentration received was used directly at volumes specified below, for each respective experiment. All experiments were conducted under strict aspectic conditions and, where necessary, 70% ethanol was sprayed at regular intervals.

The human colorectal adenocarcinoma cell line, HT29, was obtained from the European Collection of Authenticated Cell Cultures (Salisbury, UK) and cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium plus GlutaMAX™ (Gibco® by Life Technologies™, Paisley, UK) supplemented with 10% (v/v) Foetal Bovine Serum (FBS) (Sigma-Aldrich, Gillingham, UK). The photosensitizers Hypericin and Foscan were obtained from Sigma Aldrich and biolitec Pharma Ltd. (Jena, Germany) respectively. Thiazolyl Blue Tetrazolium Bromide (MTT) was obtained from Sigma Aldrich. Hypericin and Foscan stock solutions were prepared in 100% ethanol. Final concentration of ethanol in cell media for Hypericin was 0.2% for in vitro experiments and 6% ethanol per 0.5 mg kg⁻¹ injection. Final concentration of ethanol in cell media for Foscan was 0.5% for in vitro experiments and 10% ethanol per 0.5 mg kg⁻¹ injection.

Pheophorbide a was purchased from Frontier Scientific Inc. (Logan, UT, USA) and hypericin and methylene blue were purchased from Sigma-Aldrich Co. (St Louis, MA, USA). The PS solution for in vitro PDT study was prepared freshly by dissolving Pa and Hy in DMSO to make a 10 mM stock solution. It was then diluted in Tween 80 and MHB to set the desired stock solution. A serial two-fold dilution procedure was employed to obtain final working concentrations. Tween 80 and DMSO concentrations were maintained ≤ 0.1% and ≤1% (v/v), respectively, in each test group.
The bacterial strains MRSA, ATCC 43300, ATCC BAA-42, ATCC BAA-43, ATCC BAA-44, two mutant strains [AAC(6)′ APH(2)′′ and RN4220/pUL5054], five community-acquired (CA-MRSA) and five hospital-acquired MRSA (HA-MRSA) clinical strains were obtained from the Department of Microbiology, Faculty of Medicine, The Chinese University of Hong Kong.

To treat the cells, 100 or 200 nM hypericin (Sigma-Aldrich, Darmstadt, Germany) dissolved in DMSO or a vehicle of 100% dimethyl sulfoxide (DMSO, Sigma-Aldrich) was administered as the DMEM solution (DMSO < 1%) in the absence of light. After 3 h, the medium containing hypericin was removed, and a fresh complete medium was added to the cells. The cells were then treated with PDT (590 ± 10 nm light emitting diodes, 2 min, 2 J/cm², 16.7 mW/cm²) and kept in the dark for 24 h. Following this, the cells were treated with PBM induced with a light dose of 2 J/cm² (185 s with an irradiance of 10.8 mW/cm²). PBM was applied after 24 h of PDT treatment (see scheme in Figure 1A). The PBM protocol was achieved by the application of light at 808 nm (infrared diode laser, Changchun New Industries Optoelectronics Tech. Co. Ltd., Changchun, China) joined with a frontal diffuser of light (Medlight SA, Ecublens, Switzerland) to reach the illumination area (45 cm², 675 mW). The evaluation of the processes occurring in the treated cells was performed 24 h after PBM treatment (see scheme in Figure 1A).