The SWCNTs, having a diameter of 1–2 nm, a purity of > 90%, and a length of > 50 mm, were procured from AD Nanotechnology, Bengaluru, India. Chitosan (min 70%, (C6H11NO4)n), folic acid (MW 441.4 g mol−1), oxaliplatin (MW 397.3 g mol−1), N, N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydrochloride (EDC. HCl) (MW 191.7 g mol−1), nitric acid, and sulfuric acid were purchased from Research laboratories, Mumbai, India. The other chemicals used in the experimental work were of AR grade.

Purification and functionalization of single-walled CNTs

The purification process of single-walled carbon CNTs involved the use of an ultrasonic-assisted acid digestion method [37]. Initially, 0.5 g of single-walled CNTs underwent bath sonication in a 100 mL solution of 0.5 M HNO3, with this treatment lasting for 0.5 h. Subsequently, the single-walled CNTs were refluxed at 120 °C for a 24 h duration. Afterward, the solution underwent filtration, and the resulting material was rinsed with distilled water until the pH of the filtrate became neutral. The cleaned SWCNTs then underwent an additional 0.5 h bath sonication in water and were subsequently dried at 100 °C for 12 h. This purification process yielded a thin, black mat comprising purified SWCNTs.

Furthermore, SWCNTs were functionalized with briefly, 120 mL of a solution containing 96% H2SO4 and 70% HNO3 (v/v = 3:1) were taken in a beaker. Then, 30 mg of SWCNTs were added to it. At 80 °C for about 16 h, the mixture was then subjected to ultrasonic irradiation. Further, it was filtered, washed with the distilled water until a pH of 7.0 was reached, and finally dried in a vacuum to obtain functionalized SWCNTs (FSWCNTs) [38,39,40].

Preparation of chitosan-loaded system CHI-FSWCNTs

To enhance the aqueous solubility of modified COOH-SWCNTs, we employed chitosan, a biopolymer, by subjecting 20 mg of functionalized COOH-SWCNTs to ultra-sonication within a solution containing chitosan (40 mg) dissolved in 40 mL of 0.1 M aqueous sodium chloride for approximately 25 min as per procedure mentioned by Randive et al., 2023 [41]. Subsequently, the solution was maintained at room temperature and continuously stirred for 18 h using a magnetic stirrer. Chitosan in the free form was removed by washing the CHI-FSWCNTs using centrifugation for an 8000 rpm. CHI-FSWCNTs were then subjected to drying and stored properly.

Oxaliplatin loading to the CHI-FSWCNT

In the third step, 9 mg of oxaliplatin (OXA) and 3 mg of CHI-FSWCNTs were added to 6 mL of phosphate buffer solution (PBS) (pH 7.4) and stirred at ambient temperature for about 16 h. CHI-FSWCNT-OXA, thus obtained, was recovered by centrifugation with PBS until the supernatant liquid was colorless. The concentration of free drug (unbound) was measured spectrophotometrically at 520 nm.

Preparation of folic acid-loaded system CHI-FSWCNTs-OXA

The obtained CHI-FSWCNT-OXA (4 mg) in the aforesaid third step was suspended in a PBS buffer solution (8 mL; pH 7.4) containing folic acid (6 mg). 5 mg of 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl was added to facilitate solubilization of folic acid. The resulting mixture was allowed to stand at room temperature for approximately 16 h while being stirred. Finally, ultrapure was used to wash the obtained product a number of times so as to get rid of the unreacted substances. It was dried properly at ambient temperature to obtain the final nanosystem FA-CHI-FSWCNT-OXA [42].

Characterization of developed systemFTIR spectroscopy

FSWCNT, CHI-SWCNTs, OXA, and FA-CHI-FSWCNT-OXA nanocomposites were analyzed, and their spectra were determined using an FTIR spectrophotometer (Bruker Alpha Echo ATR) at the scanning range of 4,000–400 cm−1.

SEM and TEM analysis

SEM (Schottky, SU5000) and TEM analysis were used to determine the structure of the SWCNT and FA-CHI-FSWCNT-OXA nanocomposites, following the loading of oxaliplatin.

X-ray diffraction (XRD)

XRD-6000, an X-ray diffractometer (Bruker: range: D8 discover), was used to determine the XRD patterns of FA-CHI-FSWCNT-OXA. The conditions are being maintained as follows: Range—2 h, Speed—4°/min at 10°—80°, Voltage—40 kV and 30 mA, and Radiation—Cu Kα within configurations of θ–2θ).

Zeta potential and particle size analysis

The HORIBA scientific Nanopartica SZ100 was used for the determination of the size of the particle and their zeta potential; the conditions being 30 °C and an angle of detection of 90°, 0.1 mL of FA-CHI-FSWCNT-OXA 10 mg mL−1 was diluted to 10 mL with DMSO, 5 mL of this diluted sample was transferred to a cuvette, and the zeta potential was determined [43]. The particle size of the FA-CHI-FSWCNT-OXA composite was confirmed by a particle size analyzer.100 µL of FA-CHI-FSWCNT-OXA containing 10 mg/mL of composite was diluted with an appropriate volume of DMSO, and the particle size was measured.

Differential scanning calorimeter (DSC)

The changes in the relative specific heat associated with transitions in carbon NTs were recorded by DSC, which offers fruitful information related to physicochemical changes owing to exothermic as well as endothermic transitions, or any alteration in the heat capacity. Nanosystem FA-CHI-FSWCNT-OXA was characterized by the DSC analysis [44].

Thermo-gravimetric analysis (TGA)

Chemical as well as physical characters like sublimation, vaporization, absorption, adsorption, chemisorption, desorption, dehydration, redox reactions, and decomposition are obtained by TGA [45, 46]. The TGA graph represents the change in mass of carbon NTs as a function of time or temperature, indicating either a weight increase or decrease. Physicochemical properties of the FA-CHI-FSWCNT-OXA complex were characterized by the TGA.

Drug entrapment efficiency (EE)

Often, estimating drug EE involves quantifying the amount of drug present in CNTs, which was done using a method specified by Sobh et al. [47], wherein the amount of unloaded drug was determined in the clear supernatant of CNTs by centrifugation at 5000 rpm (30 min) which was then recorded as absorbance at 520 nm using an ultraviolet–visible spectrophotometer (Jasco V630, Japan). The standard plot for oxaliplatin was also determined. EE of oxaliplatin was estimated as per the equation stated below:

$$\mathrm \left(\%\right)=\frac}}}\times 100$$

(1)

In vitro drug release studies

The dialysis bag method was used to perform the in vitro drug release of oxaliplatin from FA-CHI-FSWCNT-OXA nanocomposites. The studies were carried out in a vessel that contained simulated intestinal fluid or simulated gastric fluid. Dialysis bags (Mol. Wt. cut-off 12 kDa, purchased from Sigma-Aldrich) were dipped in the buffer solution for equilibration before their use. In 3 mL of buffer saline, 500 mg of FA-CHI-FSWCNT-OXA nanocomposites were suspended, and then it was filled into the dialysis bag, which was suspended in the receptor compartment containing dissolution medium (50 mL). The media was kept closed and stirred at 100 rpm at 37 °C ± 0.5 °C temperature [32]. A sample (3 mL) was withdrawn at specific time intervals for analysis of the drug released from FA-CHI-FSWCNT-OXA nanocomposite, and every time a similar amount of buffer was replenished so as to keep up the sink conditions. The amount of drug released was noted spectrophotometrically at 520 nm [47].

Cytotoxicity assayMTT assay

Dulbecco’s modified Eagle’s medium (DMEM) that has been fortified with 10% fetal bovine serum was used to preserve the COLO320DM and HT29 cell lines [45]. The cells were added to a 96-well plate at a density of 1 × 104cells mL−1 per well and cultured for 24 h at 37 °C. During incubation, the cells were consequently exposed to 100 µg mL−1 of the oxaliplatin as a standard and FA-CHI-FSWCNT-OXA. For 24 h, the plates were incubated, and then 10 µL of MTT (thiazolyl blue tetrazolium bromide) dye (5 mg mL−1 in phosphate-buffered saline) was added per well so as to measure the cell proliferation. Further, the plates were incubated for 4 h at a temperature of 37 °C in a humidified chamber containing 5% CO2. It resulted in the formation of formazan crystals, owing to the reduction in dye by viable cells. The formed crystals in each well were dissolved by adding 200 µL of DMSO. The absorbance of the resulting solution was measured at 490 nm. The cytotoxicity of the compounds was determined using the following formula [45]:

$$\begin&\mathrm\\&\quad=\frac-\mathrm}} \times 100\end$$

(2)

SRB assay

DMEM fortified with 10% fetal bovine serum was used to maintain human COLO 320 DM and HT29 cell lines. The cells were cultured at a density of 1 × 104 cells per well in a 96-well plate, solution was added, and the culture was incubated continuously for 24 h at 37 °C. Following that the cells were exposed to 100 µg mL−1 of oxaliplatin, FA-CHI-FSWCNT-OXA and standard. Thereafter, 50 µL TCA (50%) was added to each well separately, and the plate was kept at 4 °C for 1 h. The plates were then washed with TDW (triple-distilled water) and allowed to air dry. Further, 100 µL SRB dye was added to each well and set aside for 30 min at room temperature. Lastly, the plates were washed three times with 1% acetic acid and allowed to air dry. Subsequently, 200 µL of tris buffer solution was used, and the absorbance was measured at 490 nm. The percent cytotoxicity exhibited by the compounds under study is calculated using Eq. (2) [48,49,50,51,52,53].

Apoptosis assay

Flow cytometry was used for the apoptosis study of the cancer cells using altered plasma membrane phospholipids filled with Annexin V-FITC (a lipid-loving dye). In a 24-well flat-bottom microplate equipped with cover slips, the selected cells were suspended. The cells were maintained in a CO2 incubator at 37 °C for 12 h and washed repeatedly with PBS.

After washing the cells, they were centrifuged at 500 ×g for 5 min at 4 °C. The supernatant liquid was removed, and the obtained cell pellets were resuspended in ice-cold 1X binding buffer at 1 × 105 mL−1, whereas the cold temperature was maintained by keeping the tubes in an ice bath. Thereafter, Annexin V-FITC (1 µL) and PI (5 µL) was added in each sample. All tubes were placed in an ice bath and incubated in the dark for 15 min. Finally, ice-cold 1X binding buffer (400 µL) was added and mixed gently, and labeled cells were analyzed immediately under flow cytometry with Cell Quest software (Becton Dickinson) [50].

Statistical analysis

GraphPad Prism 8 software (Windows 64-bit version 8.0.1) was used to analyze the statistical data obtained from the cytotoxicity studies. The results obtained after the performance of the tests were analyzed by one-way ANOVA with Dunnett’s post-test analysis of variance. Mean ± SEM of all the calculated values was calculated. P < 0.05, 0.01, or 0.001 were considered statistically significant. For the determination of the size of the prepared systems, ImageJ software (Java2HTML Version 1.5) was utilized.