Metformin, 5-fluorouracil, chitosan, tripolyphosphate, sodium hydroxide, and acetic acid were purchased from Lab Chemicals Private Limited, Chennai, Tamil Nadu. HTC 116 cancer cell line, MTT reagent, dimethyl sulfoxide, CCK-8 reagent, and fetal bovine serum were procured from Sigma-Aldrich.

Preparation of MET and 5-FU-nps

5-FU and MET nanospheres with chitosan as a biodegradable polymer were formulated by the ionotropic cross-linking method; chitosan was used as the polymer and tripolyphosphate as the cross-linking agent. 0.5, 1, and 1.5 w/v of chitosan solution were prepared by dissolving 0.5, 1, and 1.5 gm of chitosan in 100 ml of acetic acid (2%) v/v, followed by the addition of sodium hydroxide solution (20 wt%) to adjust the pH to 4.7. 0.25 gm of MET and 0.25 gm of 5-FU were dissolved in 100 ml of distilled water, and the stock solution was prepared. 5 ml of stock solution was slowly mixed into the chitosan solution so that the drug is integrated with the chitosan biodegradable polymer. 1% w/v of tripolyphosphate reserve liquid was incorporated very slowly into the chitosan solution with constant stirring and centrifugation at 3500 rpm for 3 h till the opaline appearance was noticed in the mixture. It was then filtered, washed, and dried [12]. Different ratios of drug and polymer combinations were used during the formulation: 1:1, 1:2, and 1:3, keeping the concentration of tripolyphosphate the same as described in Table 1.

Table 1 Formulation of NPs with different concentrations of chitosan

Formulation F3, which contains 1.5% chitosan (1:3), was selected as the best formulation based on the properties of the nanoparticles generated. When combined with oppositely charged TPP, chitosan gels create uniform spherical particles. This first rapid release, known as the “burst effect,” is driven by the fact that MET-5-FU drugs were concentrated on the surface of nanoparticles through adsorption and were readily released. Following the initial burst effect, there was a gradual, more controlled release during the release period. Drug release in alkaline media was shown to be controlled by the swelling behavior of chitosan nanoparticles in terms of pH sensitivity, which increases the diffusional path length of the drugs [13]. The formulation was optimized with the help of Box and Behnken—design expert software—version 12. The details of the optimized formulation are available from previously published article of Priya et al. [14].

Characterization of npsDrug entrapment efficiency and drug loading

The amount of drug incorporate into the biodegradable chitosan polymer is calculated by taking a measured quantity of the nanospheres. The nanospheres were pulverized, and the powdered particles were made into a solution by the addition of 10 ml of phosphate buffer, and a pH of 7.4 was maintained. This solution was stirred for 15 min and left to stand for 24 h. The supernatant was removed, and the remaining solution was filtered. The filtrate was diluted with phosphate buffer pH 7.4, and the absorbance of the sample was determined spectrophotometrically [15]. Drug entrapment efficiency is calculated from the total amount of drug encapsulated divided by the total amount of drug taken for the preparation.

Particle size analysis

The particle dimension and distribution patterns were determined with the Zeta size analyzer (Shimadzu, Japan) fitted to Wing software (version 1201). The average particle size and polydispersity index were analyzed.

FT-IR

Fourier transform infrared spectra of the sample were analyzed in the range of 400–4000 cm−1 with 4 cm−1 resolutions. The samples to be tested were mixed with potassium bromide in a ratio of 1:100 and pressed together to form a pellet. The pellet was placed on the rack and kept on the light path, and analyzed. Infrared spectra of pure drugs and drugs with polymers were recorded [16].

Differential scanning calorimetry (DSC)

The thermal analytical method was used to understand the heat transition of the drug substance in this process. The calculated quantity of the sample was placed on an open aluminum pan and subjected to high-temperature heating processed between 0 and 400 °C in the atmosphere of nitrogen for 10 min, and then, thermal transitions were recorded [17].

Scanning electron microscopy

The sample was placed on the brass stub coated with double-sided adhesive carbon tape. The stub was placed in the sample chamber and subjected to a focused electronic beam to scan the sample and study the surface characteristics of the NPs.

Optical microscopy

The sample is placed on the glass slide, allowing sufficient amount of light to pass through the sample. The magnified image is captured to study the morphological features of the formulation.

In vitro release

An USP Dissolver II-Paddle Type (Electro Lab, India) was used to evaluate the in vitro release performance of 5-FU-MET nanoparticles at 37 ± 1 °C and peddling speed of 100 rpm. An accurately weighed amount of MET and 5-FU-loaded nanoparticles equivalent to 100 mg MET and 5-FU was added to 900 mL of dissolution medium (0.1 N HCl) for 2 h, and then, dissolution in phosphate buffer (pH 7.4) was carried out. At the marked time interval, a portion of the sample, around 5 ml, was drawn periodically and filtered through a 0.45μ membrane filter. The filtered sample was subjected to analysis using a UV spectrophotometer. Each time 5 ml of sample was withdrawn, and 5 ml of phosphate buffer was replaced. The absorbance of the sample was measured at predetermined time intervals, the cumulative percentage of the drug released was measured, and the graph was plotted [18].

Kinetic study

In vitro drug dissolution kinetics was analyzed with the help of the kinetic models like zero-order, first-order, the Higuchi model, Hixson–Crowell, and the Korsmeyer–Peppas model for kinetics, were considered for the model fit. The correlation coefficient and rate constant were derived from the linear curves of regression analysis. The drug liberated from MET and 5-FU-NPs was calculated from the drug absorbance data obtained through a UV spectrophotometer. The percentage of cumulative drug released from MET and 5-FU-NPs was used to determine the diffusion exponent.

$$Q\, = \,K_}} t^\kern-.1em/ \kern-.15em\lower.25ex\hbox }$$

Q is the drug released, and KH is the Higuchi’s rate constant.

The cumulative percentage of drugs released vs. the square root of time interprets the diffusion.

Cube root CBR (Wo)-CBR (Wt) versus time interprets the drug dissolution rate constant.

Log cumulative percentage of drugs released versus log time indicates swelling and diffusion [19].

MTT assayCell proliferation assay

We conducted the cell proliferation assay using the cell counting kit-8 and the colony formation assay. HCT 116 cells were seeded onto 96-well plates and cultured for 24 h using 10% fetal bovine serum (FBS) culture medium, penicillin (100 U/mL), and streptomycin (100 g/mL) The mixture was incubated with MET (0, 1.56 µM, 3.12 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) or 5-FU (0, 1.56 µM, 3.12 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) or MET and 5-FU (0, 1.56 µM, 3.12 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) or MET and 5-FU-NPs (0, 1.56 µM, 3.12 µM, 6.25 µM, 12.5 µM, 25 µM, 50 µM, 100 µM) for 24 h and were sterilized by membrane filtration (0.2 μm) in a laminar hood. Then, the cells were treated for 4 h at 37 °C in 5% CO2 atmosphere with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). To dissolve the formazan crystals, 1 ml of dimethyl sulfoxide (DMSO) was added. The water-soluble tetrazolim dye, which is yellow in color, gets converted to purple color due to the enzyme mitochondrial reductase from the live cell. The supernatant was removed, 100 µl of propanol was added, and plates were gently shaken to solubilize the formed formazan. CCK-8 reagent (10 µl) was added to each well and again incubated for 2 h. The absorbance of the samples was measured by a microplate reader (Model No. 680XR reader Bio Rad) at a wavelength of 550 nm; the viability of the cells was then calculated [20].

Drug sensitivity assay

The effects of MET, 5-FU, MET and 5-fluorouracil, and MET and 5-FU-NPs on the HTC 116 cell line were studied to assess their cytotoxicity with the help of an MTT assay. HTC 116 colorectal cancer cells and control cells were plated in 96-well plates at a density of 105 cells/well and incubated for 24 h. The medium was removed after 24 h and replaced with fresh medium containing a measured quantity of the sample (5-FU, MET, and a combination of 5-FU and MET and MET and 5-FU-NPs) for another 48 h. 10 μL of sterile MTT dye (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, 5 mg/mL) from Sigma was added to the culture medium at a concentration of 0.5 mg/mL and incubated at 37◦C for 2 h. The formazan crystals formed were dissolved in 100 μL of dimethyl sulfoxide (DMSO) for 10 min. The absorbance of the sample was measured in a spectrometer at 550 nm using a microplate reader. The percentage inhibition and percentage viability of the cells were calculated. The dose of drug that causes 50% cell viability is considered the IC50 value of the drug [5, 19,20,21].

IC 50 calculation:

IC 50 values are calculated using the formula: \(Y = MX + C\) (from the graph).

To calculate X,

X = (Y−C)/M

Y is 50

M is slope

X is concentration

C−Y is the intercept.

Statistical analysis

All the numerical data were expressed as mean and standard deviation. Simple linear regression and multiple linear regression were used to establish the relationship between the variables. Regression coefficient was used to measure linear correlation between the variables. Statistical Package for Social Science version 29 from IBM was used to perform all statistical analysis.