Bromides

Example 5

MTT cytotoxicity assays were used to assess the cytotoxicity of RGQDs/Nd-GQDs/Tm-GQDs. HeLa cells were plated in a 96-well plate with 5000 cells per well (100 μL/well) and kept in an incubator overnight at 37.1° C. while maintaining the CO₂/air ratio of 1:19. After 24 h of incubation, the samples were added into each well at different concentrations for different materials (0 to 70 μg/mL, 1 mg/ml, 0.25 mg/ml for RGQDs, Nd-GQDs, Tm-GQDs, respectively). After 24 h of incubation, the medium was replaced by 100 μL of 1 mg/mL thiazolyl blue tetrazolium bromide. After 4 h of further incubation, MTT (3-(4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was replaced with 100 μL of DMSO (dimethyl sulfoxide) to solubilize the precipitation. Reduction in MTT influences the metabolic activity of living cells, which can be assessed with absorbance measurements because living cells metabolize the MTT and form a highly absorbing purple colored byproduct known as formazan. The absorbance (essentially the cell viability) of the final sample was measured at 540 nm wavelength using the FLUOstar Omega microplate reader.

Example 7

The MTT Cell Proliferation assay determines cell survival following apple stem cell extract treatment. The purpose was to evaluate the potential anti-tumor activity of apple stem cell extracts as well as to evaluate the dose-dependent cell cytotoxicity.

Principle: Treated cells are exposed to 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTT enters living cells and passes into the mitochondria where it is reduced by mitochondrial succinate dehydrogenase to an insoluble, colored (dark purple) formazan product. The cells are then solubilized with DMSO and the released, solubilized formazan is measured spectrophotometrically. The MTT assay measures cell viability based on the generation of reducing equivalents. Reduction of MTT only occurs in metabolically active cells, so the level of activity is a measure of the viability of the cells. The percentage cell viability is calculated against untreated cells.

Method: A549 and NCI-H520 lung cancer cell lines and L132 lung epithelial cell line were used to determine the plant stem cell treatment tumor-specific cytotoxicity. The cell lines were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml) in a 5% CO₂ at 37 Celsius. Cells were seeded at 5×10³ cells/well in 96-well plates and incubated for 48 hours. Triplicates of eight concentrations of the apple stem cell extract were added to the media and cells were incubated for 24 hours. This was followed by removal of media and subsequent washing with the phosphate saline solution. Cell proliferation was measured using the MTT Cell Proliferation Kit I (Boehringer Mannheim, Indianapolis, IN) New medium containing 50 μl of MTT solution (5 mg/ml) was added to each well and cultures were incubated a further 4 hours. Following this incubation, DMSO was added and the cell viability was determined by the absorbance at 570 nm by a microplate reader.

In order to determine the effectiveness of apple stem cell extracts as an anti-tumor biological agent, an MTT assay was carried out and IC50 values were calculated. IC50 is the half maximal inhibitory function concentration of a drug or compound required to inhibit a biological process. The measured process is cell death.

Results: ASC-Treated Human Lung Adenocarcinoma Cell Line A549.

Results: ASC-Treated Human Squamous Carcinoma Cell Line NCI-H520.

Results: ASC-treated Lung Epithelial Cell Line L132.

Summary Results: Cytotoxicity of Apple Stem Cell Extracts.

Apple stem cell extracts killed lung cancer cells lines A549 and NCI-H520 at relatively low doses: IC50s were 12.58 and 10.21 μg/ml respectively as compared to 127.46 μg/ml for the lung epithelial cell line L132. Near complete anti-tumor activity was seen at a dose of 250 μg/ml in both the lung cancer cell lines. This same dose spared more than one half of the L132 cells. See Tables 7-10. The data revealed that apple stem cell extract is cytotoxic to lung cancer cells while sparing lung epithelial cells. FIG. 6 shows a graphical representation of cytotoxicity activity of apple stem cell extracts on lung tumor cell lines A549, NCIH520 and on L132 lung epithelial cell line (marked “Normal”). The γ-axis is the mean % of cells killed by the indicated treatment compared to unexposed cells. The difference in cytotoxicity levels was statistically significant at p≤05.

Example 9

The experiment of Example 7 was repeated substituting other plant materials for ASC. Plant stem cell materials included Dandelion Root Extract (DRE), Aloe Vera Juice (AVJ), Apple Fiber Powder (AFP), Ginkgo Leaf Extract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells (OSC) as described in Examples 1 and 2. The concentrations of plant materials used were nominally 250, 100, 50, 25, 6.25, 3.125, 1.562, and 0.781 μg/mL. These materials were tested only for cells the human lung epithelial cell line L132 (as a proxy for normal epithelial cells) and for cells of the human lung adenocarcinoma cell line A549 (as a proxy for lung cancer cells).

A549 cells lung cancer cell line cytotoxicity results for each of the treatment materials.

DRE-Treated Lung Cancer Cell Line A549 Cells.

AVJ-Treated Lung Cancer Cell line A549 Cells.

AFP-Treated Lung Cancer Cell line A549 Cells.

GLE-treated Lung Cancer Cell line A549 Cells.

LSC-treated lung cancer cell lines A549 cells.

OSC-treated Lung Cancer Cell line A549 Cells.

L132 cells (“normal” lung epithelial cell line) cytotoxicity results for each of the treatment materials.

DRE-Treated Lung Epithelial Cell Line L132 cells.

AVJ-Treated Lung Epithelial Cell Line L132 cells.

AFP-Treated Lung Epithelial Cell Line L132 cells.

GLE-Treated Lung Epithelial Cell Line L132 cells.

LSC-Treated Lung Epithelial Cell Line L132 cells.

OSC-Treated Lung Epithelial Cell Line L132 cells.

Calculated values.

Example 5

In some embodiments, the disclosed ECP has a formula of

[Figure (not displayed)]

The ECP-5 is synthesized by preparing a naphthalene-containing reaction unit and then polymerizing it with an AcDOT unit. The detail method includes the following steps:

Step 5-1: preparing naphthalene-containing reaction unit (compound 10) by two steps.

[Figure (not displayed)]

To a solution of compound 11 in dichloromethane was added dropwise a solution of bromine in dichloromethane over 15 minutes at −78° C. The reaction mixture is stirred for 2 hours at −78° C. and then warmed gradually to room temperature and stay at room temperature for an additional 2 hours. The excess bromine was quenched by saturated aqueous sodium sulfite solution and stirred for 2 hours at room temperature. After extraction with dichloromethane, the combined organic layer was washed with brine, dried over sodium sulfate, and concentrated in vacuum.

[Figure (not displayed)]

Compound 12 is dissolved in DMF under N₂, K₂CO₃ is added to the solution, and the reaction mixture is stirred for 15 minutes, after which 2-ethylexyl bromide is added. The reaction mixture is stirred at 100° C. overnight. The reaction is stopped and cooled down to room temperature. The solvent is removed in vacuum, and the residue is dissolved in diethyl ether. The organic phase is washed with water, and the aqueous phases are extracted with ethyl acetate. The combined organic phases are dried by vacuum.

Step 5-2: polymerization: The polymerization method is similar to that in step 1-1, only differs on the reaction units. The reaction units here are the naphthalene-containing reaction unit (compound 10) and AcDOT (compound 8).