Materials

The HA was bought from Bangalore Fine Chem, Bangalore (pure powder, MW 800 KDA). Sigma-Aldrich was used to get ADH (98% purity, synthesis grade, MW 174.20) and EDC (> 98% purity, MW 191.7). From Hyderabad's Dr. Reddy's lab, PAC (MW 853) was obtained. The citric acid (MW 192), sodium hydroxide (NaOH), and hydrochloric acid (HCl) were purchased from Research-Lab Fine Chem Industries, Mumbai, and were of analytical quality. Both 3-(4, 5-dimethylthiazol-2-yl)−2, 5-diphenyltetrazolium bromide (MTT) solution and 4, 6-diamidino-3-phenylindole (DAPI) were purchased from Sigma-Aldrich. FBS came from Gibco and Antibiotic–Antimycotic 100X solution from Thermo Fisher Scientific. The use of DMEM with a high glucose content as well as other reagents and solvents was done without further purification because they were of an analytical position.

Synthesis of GQDs

By using a bottom-up approach, GQDs were produced according to a previously described methodology by Bansal et al. [30]. In a nutshell, 2 g of citric acid was heated in a muffle furnace to 160 °C for 15 min, or until the acid's color changed to orange. Additionally, 100 mL of 1.5 M NaOH solution was added dropwise beneath vigorous stirring to the pyrolyzed citric acid. The pH of the bright yellow GQDs solution was raised to 7 using 1N HCl solution. After that, the substance was freeze-dried and kept in vials for further use.

Synthesis of HA-ADH conjugation

The study's methodology was somewhat altered to focus on direct HA-ADH improvement instead of ethanol precipitation from the HA-ADH solution. In a nutshell, 12 mL of water and 60 mg of HA were combined to create a 5 mg/mL HA solution. This solution was supplemented with 1.1 g of ADH in solid form, which was then mixed for 30 min. The pH of the reaction was then increased by 1N HCl to 4.8. After that, 0.1 g of solid EDC was added. Then, 1N HCl was added to the mixture to keep the pH at 4.8. After 2 h, the process was stopped by raising the pH to 7.0 using 1N NaOH. After being transferred to prewashed dialysis tubing, the reaction mixture was dialyzed for a full day. The powdered form of HA-ADH was obtained via freeze-drying [31].

Synthesis of HA-ADH-GQDs

100 mg of GQDs was dissolved in 10 mL of pH 7.4 (phosphate buffer saline) PBS in a glass bottle. In the other bottle, 100 mg of HA-ADH was dissolved in 10 mL of pH 7.4 PBS. The HA-ADH solution and GQD solution were then combined and allowed to mingle for 24 h in the dark. Finally, after dialysis, the solution was filtered and then freeze-dried to create the HA-ADH-GQDs powder.

Loading on HA-ADH-GQDs with PAC

The PAC was loaded by mixing 30 mL of HA-ADH-GQDs (0.5 mg/mL) pH 7.4 PBS solution for 24 h in the dark at room temperature with 1.0 mL of PAC (1 mg/mL). To get HA-ADH-GQDs loading with PAC (PAC@HA-ADH-GQDs), the produced suspension was dialyzed continuously against PBS buffer for 24 h to remove unbound PAC. The PAC@HA-ADH-GQDs were then retrieved and freeze-dried to generate the PAC@HA-ADH-GQDs powder. Equation 1 is used to calculate the % loading of PAC using UV–visible spectroscopy. For this, the absorbance at 230 nm was measured using UV–visible spectroscopy (Shimadzu UV 1800) after 10 mg of PAC@HA-ADH-GQDs nanocomposite was suspended in 10 mL of pH 7.4 PBS. The following described process was used to create the PAC calibration curve to estimate % loading efficiency [32].

Ten milligrams of PAC was weighed out correctly and dissolved in 30 mL of methanol. Then, to prepare a stock solution of 100 µg/mL, the volume was increased to 100 mL using a methanol: PBS (30:70) mixture. A mixture of methanol and PBS (30:70) was used to create the bulk after aliquots of the 100 µg/mL stock withdrawal solution were transferred to separate 10 mL volumetric flasks to create the final PAC concentrations. The absorbance was measured at 230 nm using a 30:70 methanol: PBS combination as a blank and 3 mL of methanol up to 10 mL.

$$\% Drug\, loading\, efficiency = \frac \right)}} X 100$$

(1)

PAC drug release profile

HA-ADH-GQDs nanocomposite's PAC discharge release mechanism was computed in PBS at pH 7.4 and pH 5 at 37 °C in a dialysis bag. To begin with, 10 mg of PAC@HA-ADH-GQDs nanocomposite was dissolved in 10 mL of pH 7.4 PBS to create the stock solution. From that, 3 mL of the solution was put in the dialysis bag and suspended in 30 mL of pH 7.4 PBS and pH 5. Finally, samples were placed in a magnetic stirrer and heated to 37 °C for the duration of the experiment. Four milliliters of solution was removed from an external buffer to assess the concentration of PAC discharged, and the amount of drug discharge was determined using a UV spectrophotometer's acquired calibration curve at 230 nm [33]. It was discovered that hydrophobic interactions and hydrogen bonding are mostly responsible for how PAC gets adsorbed on HA-ADH-GQDs. As a result, in an acidic environment, contact decreases due to the presence of H species, and PAC discharge begins outside of HA-ADH-GQDs.

pH stability testing

According to the established ICH guidelines, stability tests for PAC-loaded HA-ADH-GQDs NPs were carried out. The pH stability of the PAC@HA-ADH-GQDs nanocomposite sample was examined in pH 7.4 PBS and pH 5. For this, 10 mg of PAC@HA-ADH-GQDs nanocomposite was added to 25 mL each of pH 7.4 PBS and pH 5, which was then placed in a clean, transparent container with the appropriate labeling. The PAC@HA-ADH-GQDs pH 7.4 PBS and PAC@HA-ADH-GQDs pH 5 samples were then transported to a stability chamber, where they were kept at 25 ± 3 °C and 55% ± 4% RH for three months. Each month, samples were analyzed for pH, physical appearance, and medication content [34].

In vitro cytotoxicity

The human breast cancer cell line MCF7 was cultured for 24 h at 37 °C in 5% CO2 and 1 × 104 cells/mL of culture media. Then, cells were seeded on microplates at a concentration of (70 μL) 104 cells/well in 100 μL of culture medium and 100 μL of a sample of the LM series at (10, 30, and 100 μg/mL, respectively) (tissue culture grade also 96 wells). After that, cell line and DMSO (0.2% in PBS) were used to cultivate control wells. Each sample was raised in three separate cultures. Additionally, controls were kept to monitor the fraction of live cells following culture as well as the survival of the control cell. Additionally, cell cultures were grown for 24 h in a CO2 incubator at 37 °C and 5% CO2 (Thermo Scientific BB150). Following incubation, the medium was completely removed and replaced with 20 μL of MTT reagent (5 mg/min PBS). Cells were nourished for 4 h at 37 °C in a CO2 incubator after MTT accumulation. The wells were then examined under a microscope to check for the formation of formazan crystals. Yellowish MTT was then reduced to dark formazan solely by functional cells. After completely removing the medium, 200 μL of DMSO was added and left for 10 min before being hatched at 37 °C and wrapped in aluminum foil (Reg. No.−18,313,003,120,315,669). By measuring each sample's absorbance using a microplate reader (Benesphera E21) at 550 nm, triplicate samples of GQDs, GQDs-HA-ADH, and PAC@HA-ADH-GQDs were examined at various concentrations like 10 µg/mL, 40 µg/mL and 100 µg/mL.

Cell imaging study

MCF7 cells were cultivated in 96-well plates (1 × 104 cells/mL) and incubated for 24 h to study the cell imaging of GQDs and PAC@HA-ADH-GQDs. Cells were preserved using 1 mg/mL GQDs, and PAC@HA-ADH-GQDs after a 24-h incubation period. A further 4 h of incubation was given to the cells. After that, cells were stained for 5 min with a 2 μg/mL solution of 4, 6-diamidino-3-phenylindole (DAPI, Sigma-Aldrich) and then repeatedly rinsed with PBS to remove any unengaged cells. Finally, an ELISA reader carried out the imaging studies of GQDs and PAC@HA-ADH-GQDs at 100 µg/mL concentration.

Cellular uptake study

2 × 105 cells per mL of cultured cells were used, and the plate was incubated at 37 °C in a CO2 incubator overnight. In 2 mL of culture medium, the cells were treated with the suitable concentration of the experimental substances and the controls and then incubated further for 48 h. The cells were then harvested into 2-mL Eppendorf tubes after the end of the treatment. Afterward, the cells were stained for 10 min with 200 µL staining solution, and subsequently, the staining solution was removed and washed with PBS to remove excess dye. Before imaging, 50 µL of the cell suspension was cautiously loaded onto the glass slide and mounted there via a drop of mounting medium. Use a filter cube and a fluorescence microscope to observe under conditions where EtBr (Ethidium bromide) is excited at 560/40 nm and emitted at 645/75 nm, and acridine orange is excited at 470/40 nm and emitted at 525/50 nm. CLSM (Confocal laser scanning microscopy) Image J Software version 1.48 was used to overlay the images of Control (52 µg/mL), b) Std. (52 µg/mL), c) GQDs (11 µg/mL) and d) PAC@HA-ADH-GQDs (10 µg/mL).

Characterization of synthesized NTCs

With the aid of the Shimadzu UV 1800, the UV–visible spectrum of PAC and PAC@HA-ADH-GQDs was put together. Using a scanning electron microscope (SEM; Jeol 6390LA/OXFORD XMX N) and a high-resolution transmission electron microscope (HR-TEM; Jeol/JEM2100), the morphology of synthetic GQDs was studied. 1H-NMR (Jeol Japan/ECZR series 600 MHz) confirmation of the synthesized HA-ADH conjugation was made. Zetasizer was used to obtain the average diameter and particle size distribution of NTCs (Malvern). To study their chemical characteristics, FTIR curves of PAC and PAC@HA-ADH-GQDs were performed (Shimadzu IRAffinity-1S). To evaluate the amount of PAC released from HA-ADH-GQDs NPs, samples of the PAC@HA-ADH-GQDs nanocomposite were taken in pH 7.4 PBS and pH 5, and the absorbance was then measured using a UV–visible double-beam spectrophotometer. The stability studies were conducted at various pH values to estimate the conjugates of PAC@HA-ADH-GQDs. GQDs, GQDs-HA-ADH, and PAC@HA-ADH-GQD samples were subjected to the MTT assay, which was performed on a microplate reader (Benesphera E21). The cell pictures of the GQD and PAC@HA-ADH-GQD samples were taken using an inverted phase microscope. Using the Image J Software version 1.48 for CLSM (Carl Zeiss, Germany), the pictures of the control, standard, GQDs, and PAC@HA-ADH-GQDs were superimposed.