{'fraction_non_alphanumeric': 0.04040542391453226, 'fraction_numerical': 0.01657307218189289, 'mean_word_length': 4.5954022988505745, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Polyethylene Glycol-Encapsulated Histone Deacetylase Inhibitor Drug-Composite Nanoparticles for Combination Therapy with Artesunate

Combination drug therapy has become an effective clinical practice for cancer treatment because of low cytotoxicity by the synergistic effect of each medicine. Luminescent Au nanoclusters (Au NCs) were formulated into spherical polyethylene glycol (PEG)–Au NC-encapsulated drug–sodium butyrate (NaB) composite nanoparticles (PEG–Au NC–NaB-NPs) in the presence of PEG and NaB. Their effect on cancer cells was investigated using bio imaging, unravelling the mechanism of the endocytosis pathway and combination therapeutic interventions with a plant-based antimalarial drug artesunate (ART). PEG–Au NC–NaB-NPs showed bright red luminescence in the lysosomal compartment of the cells upon uptake predominantly through a caveolae-mediated pathway. Combination of PEG–Au NC–NaB-NPs with ART displayed enhanced therapeutic activity at a reduced dose compared to its individual doses and revealed heightened synergistic activity as identified from the combination index. The mechanism of synergism revealed elevated generation of reactive oxygen species with both NaB and ART, which disrupts mitochondrial membrane potential as evident from JC-1 staining. Remarkably, the histone deacetylase (HDAC) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay enlightened the role of NaB and ART in HDAC inhibition and DNA fragmentation, respectively. Thus, induction of apoptosis with the synergistic effect of both NaB and ART with its meticulous mechanism makes it a promising tool for combinational cancer therapy. In vivo activity of the NPs was evaluated on Daltons lymphoma ascites bearing mice, which exhibited significant reduction of tumor volume and viable tumor cells with a prolonged life span.

7AAD kit used for apoptosis and APO-DIRECT™ Kit i.e., for TUNEL assay were acquired from BD lifescience. For lysosomal staining, cytopainter green was used, which was purchased from Abcam. For all experiments, Milli-Q grade water (18.2 MΩ cm) was used.
HeLa (human cervical carcinoma) cells were procured from National Centre for Cell Sciences, India (NCCS) Pune.

Quantum Yield Measurements:
The fluorescence quantum yield of as-synthesized PEG-Au NC-NAB-NPs was determined with quinine sulphate as reference sample where quinine sulphate was dissolved in 0.1 M H 2 SO 4 . To calculate the quantum yield of PEG-AuNC-NAB-NPs the following equation has been used: Where, QY S = quantum yield of as synthesized PEG-AuNC-NAB-NPs (sample) QY R = quantum yield of quinine sulphate, which was dissolved in 0.1 M H 2 SO 4 (reference).
The standard quantum yield of the reference (QY R ) is 0.54. A S = area under the emission spectrum of PEG-AuNC-NaB-NPs obtained at λ ex = 320 nm at slit-width = 2 nm A R = area under the emission spectrum of reference Abs R = absorbance of the reference Abs S = absorbance of the sample ƞ S = refractive index of solvent of sample

Determination of Reactive Oxygen Species Generation with FACS
The reactive oxygen species generated on treatment with Au NCs, NaB, PEG Au NC-NaB-NPs, ART and PEG-AuNC-NaB-NPs-ART were investigated in CytoFLEX flow cytometer (Beckman Coulter) by staining the samples with 2,7-dichlorofluoresceindiacetate (DCFH-DA; Sigma-Aldrich, USA). For this 10 × 10 3 cells/well were seeded in 6 well plates and were allowed to grow overnight. After acquiring required confluency, the cells were treated with IC 50 dose of Au NCs, NaB, PEG Au NC-NaB-NPs, ART and PEG-AuNC-NaB-NPs-ART for 3 h. Thereafter, the respective wells along with the control were incubated with 10 μM of DCFH-DA at 37 °C for 30 min. DCFH-DA, which is a flurogenic dye diffuses into the cell through plasma membrane and is converted to a non-fluorescent compound DCFH by cellular esterases. The DCFH in presence of ROS generated by the cell is oxidized into highly fluorescent compound 2', 7' -dichlorofluorescein (DCF) having green fluorescence (λ ex =488 nm and λ em =530 nm). Thus the obtained green fluorescence was analyzed in FITC channel (530/30nm), which corresponds to green emission. For this the cells were collected after 30 min treatment and were analyzed using CytoFLEX flow cytometer (Beckman Coulter).

Cell Cycle Analysis
Propidium iodide (PI) based cell cycle analysis for DNA content measurement was carried out in CytoFLEX flow cytometer (Beckman Coulter), which distinguishes cells in different phases of the cell cycle. Here PI stains DNA quantitatively after permeabilisation. Cells (10 × 10 3 cells / well) were seeded in a 6 well plate and treatment with IC 50 dose of Au NCs, NaB, PEG Au NC-NaB-NPs, ART and PEG-AuNC-NaB-NPs-ART were given for 48 h.
Thereafter cells were collected and fixed with ice cold (1 mL) 70% ethanol. Ethanol was be added slowly while vortexing and cells were then stored at -20 °C for 1 h. Then 70% ethanol was removed by centrifugation by discarding the supernatant and the pellet was redispersed in PBS followed by incubation in 0.4 mg/mL RNase solution for 1 h at 37 °C. At last cells were incubated with PI (10 μg/mL) for 30 min under dark condition and thereafter analysis was carried in PE-A channel (band-pass filter, 585/42 nm) of CytoFLEX flow cytometer (Beckman Coulter) using an excitation wavelength of 488 nm. The ModFit LT software 5.0 was used for data analysis.

PE Annexin V-7-AAD Apoptosis Detection Assay
For detection of early and late apoptotic cells, PE Annexin V detection kit (BD Biosciences) in conjunction with 7-amino-actinomycin (7-AAD) dye was used. In a similar way, 10×10 3 cells were seeded in 6 well plates and were allowed to grow for 24 h. This was followed by treatment with Au NCs, Sodium Butyrate, PEG Au NC-NaB-NPs, ART and PEG-AuNC-NaB-NPs-ART for 48 h. In dead cells, because of membrane damage, 7-AAD is permeable as opposed to intact membrane in viable cells. Thus viable cells are both PE Annexin V and 7-AAD negative; early apoptotic cells are PE Annexin V positive and 7-AAD negative and dead cells are both PE Annexin V and 7-AAD positive. The samples were prepared based on the protocol provided by the company (BD Lifescience) and were analysed in CytoFLEX flow cytometer (Beckman Coulter).           Figure S12. FACS analysis of ROS production evaluated by DCF fluorescence in HeLa cells investigated in FITC-H channel, which corresponds to the green emission of the DCF. Cells were treated with IC 50 dosage of Au NCs, NaB, ART, PEG-AuNC-NaB-NPs and PEG-AuNC-NaB-NPs-ART, which exhibited prominent shifts as compared to that of control cells.  magnification.

Measurement of Mean Survival Time (MST) & Increase in Life Span % (ILS):
To determine the effect of drug treatment on mortality rate of the cancer (DLA) mice, five mice from each treated groups were monitored for 50 days. Kaplan-Meier curve was established to represent the survivability of mice following drug treatment. MST and % ILS determined with the following formulas: Figure S16. Confocal microscopic fluorescence images of DLA cells after 4 h of incubation demonstrating the uptake of Peg-Au NC-NaB-NPs. The excitation wavelength was 405 nm. All the results were expressed in mean ± S.D. * p < 0.05 in comparison of drug treated groups with untreated group.