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5-FU 99% purchased from (Cas
No. 51.21.9) Sigma-Aldrich, India, ?-tocopherol was purchased from
TCI chemicals (India) Pvt. Ltd (Cas No. 59-02-8). PLGA purchased from Sigma-Aldrich,
India (Product No. 808482-5G), SCC15 cell lines procured from National Centre for Cell Sciences, Pune,
India. The other entire chemicals (analytical grade) were purchased from
Sigma-Aldrich, India. All the experiments were performed in triplets
(n=3).

Preparation of targeted ?-T-FU-PLGA-NPs & Non-targeted 5-FU-PLGA-NPs

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5-FU was conjugated to PLGA by the ionic
cross-linking and ?-tocopherol use as a functionalized surface moiety for the
preparation of ?-T-FU-PLGA nanoparticles. PLGA 34.50 mg was dissolved in 10 ml acetic acid 1% w/v, pH was maintained at
4.8. The drug 5-FU was added to that
solution. The solution was added in 0.5% polyvinyl
alcohol (PVA) solution and allow for magnetic stirring for one hour. The
5-FU-PLGA solution was allowed to ultrasonicated
for 12 minutes at 20% amplitude to facilitate the solubility and retrieved a
homogeneous amalgamation followed by
washing with deionized water, then lyophilized and stored at 4 °C.

Surface functionalized of ?-Tocopherol as targeted moiety on 5-FU-PLGA
nanoparticles

30.25 mg of ?-tocopherol added in pH 7.4 phosphate
buffer saline (PBS) and  Subsequently,
17.5 ml 0.1% (w/v) 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide aqueous solution was added dropwise, under low velocity magnetic stirring
condition about two hours to form cross-links. The formations of nanoparticles
have been formed impulsively under the carbodiimide
reaction. The prepared 5-FU-PLGA nanoparticles were
activated by adding in 7.4 PBS solutions and 250 ul of N-hydroxysuccinimide
(NHS, 1 mg/ml) under magnetic stirring for 3 hours after that un-reacted
chemical have been washed with PBS (phosphate buffer saline) buffer. Both the
solution was added under magnetic
stirring further for 3 hours followed by
overnight incubation and ultra-sonicated for 15 min and pellets were collected
after washed with PBS. Eventually, the targeted ?-T-FU-PLGA nanoparticles were
obtained and used for furthermore experiments.

Characterization of ?-T-FU-PLGA/5-FU-PLGA NPs

The particle size, zeta potential and PDI of
targeted, ?-T-FU-PLGA & Non-targeted, 5-FU-PLGA nanoparticles were
optimized through the instrument Malvern
Zetasizer ver.7.12, (Malvern Instrument,
U.K.), and morphology of the nanoparticles obtained through scanning electron
microscope (SEM), (Quanta 450 FEG, Netherlands).

In-vitro drug release system and
drug entrapment efficiency

To study the release system and entrapment
efficiency of 5-FU-PLGA/?-tocopherol-FU-PLGA nanoparticles, a standard curve has been plotted between 5-FU
concentration (µg/ml) and absorbance (nm). The absorbance of the solution of
5-FU was established by the ultra-violet (UV) spectrophotometer (PerkinElmer,
Lambda 25, US) at absorption maxima 267 nm. The ?-tocopherol-FU-PLGA/5-FU-PLGA
nanoparticles were accommodated into a dialysis
bag and engrossed at different pH 7.4 & pH 4.5 in PBS. The dialysis bag
(Sigma Aldrich, India) procured as per the protocol. The USP dissolution
appliance grade 1 basket type was used to
perform the analysis, the speed of the
apparatus 100 rpm, 37 ºC. The dialysis bag was poured into the dissolution
solution (gastric fluid maintained pH 7.4 & 4.5), and the 5 ml supernatant
at definite time interval in hours (0, 20, 40, 60, 80, 120, 160) was withdrawn and further analyzed for drug
content through established standard calibration curve of the 5-FU solution using UV visible spectrophotometer
and in-vitro drug release were calculated through given formula. The entrapment
efficiency, also quantify with
standard curve plotted for 5-FU solution, the weigh sample of
5-FU-PLGA/?-tocopherol-FU-PLGA nanoparticles undergone at high rpm (rotation
per minutes) centrifugation at approximately 12000 rpm for 45 minutes and
collect the supernatant and filter with 0.2µm membrane filter, and further
absorbance monitored, and percentage calculated through given formula.

In-vitro
drug release (%): Concentration of drug at different time interval/total amount of drug × 100.

Entrapment
efficiency (%): Total amount of drug – the free drug in supernatant/total
amount of drug × 100.

Cell Culture

Human tongue squamous
cell carcinoma cells, SCC15, as oral
cancer cell lines, were procured from
National Centre for Cell Sciences, Pune, India, and cultured in a suitable
medium (DMEM/F12) and supplemented with 10% heat-inactivated
fetal bovine serum followed by addition of 1% antibiotic cocktail of
streptomycin and penicillin. Cells maintained in a standard humidified
incubator supplied with 5% CO2, 95% air at 37±0.50C.

Cytotoxicity of ?-Tocopherol-FU-PLGA/5-FU-PLGA nanoparticles
by MTT assay

SCC15
cells were seeded with the density of 1×104
cells into 96 well plates and acquiesce to abide by 24
hours. The SCC15 cells were exhibited to 10µL of ?-tocopherol-FU-PLGA/5-FU-PLGA
nanoparticles at predetermined
time intervals (24, 48, 72 hours) in a different dose, MTT (methyl thiazolyl tetrazolium)
(0.2mg/mL) was composite to all the well plate and sustenance for 4 to 6 hours.
250 µL, DMSO was mixed after the removal of medium and further vibrated for 12
minutes.

Cytotoxicity of ?-Tocopherol-FU-PLGA/5-FU-PLGA
nanoparticles in drug-resistant SCC15
cell lines

The drug-resistant
SCC15 cell lines were placed in into 96 well plates with density of 1×104 cells per plates for
24 hours, centrifuged and procured, the drug-resistant cells were again incubated different drug
concentration of ?-tocopherol-FU-PLGA/ 5-FU-PLGA nanoparticles at dose 0, 0.25,
1.50, 3.0, 4.5, 6.0, 7.5 µg/ml, MTT assay was performed to optimized the
cytotoxicity of the SCC15.

Therapeutic productivity of prepared nanoparticles
against SCC15

The therapeutic productivity of targeted
?-tocopherol-FU-PLGA and non-targeted 5-FU-PLGA nanoparticles as
anti-proliferating agent established against SCC15 cell lines through MTT
method. The malignant calls, SCC15 were seeded in 96 well plates at a density
of 5×103 cells
per plates for overnight incubation, and the cells were treated with different
concentration 1.0mg/ml, 0.5mg/ml & 0.1mg/ml and the anti-proliferating
effect of the ?-tocopherol-FU-PLGA/5-FU-PLGA nanoparticles was examined. The cell viability of the
nanoparticles was quantified in 96 hours
and indirectly the cytotoxicity effects of the targeted & non-targeted
nanoparticles. 

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