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  • br Materials and methods br The

    2022-04-26


    2. Materials and methods
    The low molecular weight chitosan (MW = 50–190 KDa, DD 75–85%), sodium tripolyphosphate (TPP), doxorubicin (DOX) and chlorin e6 (Ce6) were obtained from Sigma-Aldrich. Human Adult der-mal fibroblasts (HADF) and MCF-7 breast cancer FLAG tag Peptide were purchased from Himedia and NCCS (Pune) respectively. Dulbecco's modified ea-gles medium (DMEM), fetal calf serum (FCS), penicillin, streptomycin, gentamycin and amphotericin B were purchased from Himedia Pvt. 
    Ltd. All other necessary chemicals are purchased from Sigma-Aldrich. Experiments were carried on aqueous medium at 25 °C under ultrasonic stirring.
    2.2. Preparation of Ce6-CSNPs and Ce6-CSNPs-DOX
    The CSNPs was prepared by adding known amount of TPP with CS solution under constant ultrasonic stirring and by slow drop-wise addi-tion by previous reported method [38]. The pH was maintained at pH 4 by adding 0.1 N HCl. The dispersion was stirred for 4 h and then stored overnight at 4 °C. Water and 0.1 N HCl were added to the dispersion to obtain a 0.05–0.1% content of CSNPs at a pH of 4.5 formulations.
    The Ce6 was dissolved in water at a concentration of 0.01% w/v. The pH was adjusted to at 6.5 and the solution was filtered. Then, the Ce6 so-lution was added slowly and drop-wise to the CSNPs dispersion under constant stirring to obtain a final volume ratio of 1:2 V. Stirring was con-tinued for 2 h prior to the surface decoration process. Nanoparticles those obtained after centrifugation at 10,000 ×g for 10 min. The super-natant was discarded, and the pellet was resuspended in the deionized water.
    For DOX encapsulation, the DOX was added into the TPP solution prior to Ce6-CSNPs formation. The DOX loadings were 20% with respect to the total amount of CS used for nanoparticles preparation.
    2.3. Characterization of Ce6-CSNPs
    The size distribution of Ce6-CSNPs was determined using dynamic light scattering (DLS Malvern MA1114862, Zetasizer) analysis. The structural elucidation and diffraction characteristics were determined using FT-IR (PerkinElmer 1640 FT-IR Spectrometer) and XRD (Rigaku X-ray diffractometer) spectroscopy respectively. The IR spectra were re-corded as an average of 50 scans. All spectra were recorded from 600 to 4000 cm−1 with a resolution of 4 cm−1. The crystalline and amorphous characteristics were evaluated by XRD. The X-ray generator was oper-ated at 40 KV tube voltages and 40 mA of tube current, using the Kα lines of copper as the radiation source. The scanning angle ranged from 5 to 100 °C for 45 min in step scan mode (step width 3°/min).
    The size and morphological characteristics of this nanoparticle were confirmed by scanning electron microscopy (Carl Zeiss Eigma-FESEM, Xmax Oxford Instruments) analysis. For this, each sample was prepared on a 300-mesh copper grid coated with carbon at a concentration of 1 mg/ml in distilled water. The surface morphology of this nanoparticle was characterized using AFM (Bruker Nanoscope analysis) which was used to image surface structures and to measure surface forces.
    2.4. Photochemical characterization
    2.4.1. Absorbance and emission spectra
    The physical loading efficiency of Ce6 in CSNPs was quantified using UV–Vis absorbance. The Ce6 in CSNPs was quantified by using UV–Vis absorbance (PerkinElmer Lambda 750 spectrophotometer). The DMSO/water (1:10 ratios) solution was added to the nanoparticles to make a clear solution. The Ce6 concentration was determined by mea-suring absorbance at 405 nm and referring to a standard curve of free Ce6 in the same DMSO/water solution.
    The Ce6 in the CSNPs were quantified using emission spectra (Jobin Yvon Spex Fluoromax 4). Emission and excitation spectra were obtained from 550 nm to 800 nm and 300 nm to 650 nm in 1 nm steps, respec-tively. Spectra were recorded for Ce6-CSNPs dissolved in DMSO/water solution which compared with free Ce6 solution.
    2.4.2. Singlet oxygen generation and photostability
    The generation of singlet oxygen was determined using p-nitroso-N, N ′-dimethylaniline (RNO) as an indicator for photo-induced singlet oxygen with histidine as the singlet oxygen trap. Photostability experiment was made using a halogen lamp (Optel) with wavelength 630 nm long-pass
    filter, while the light was delivered to the sample through a fiber optic cable. Free Ce6 and Ce6-CSNPs were irradiated. The study was conducted for different irradiation times from 0 min to 30 min, which corresponded to doses rage from 2.28 J/cm2 to 45.6 J/cm2. After each exposure, the ab-sorption spectra were collected. These irradiation conditions correspond to measurements carried out in vitro. Study was performed for Ce6 and Ce6-CSNPs dissolved in DMSO/water.