**3. Physicochemical Characterization of APG-Loaded PEGylated-CNPs**

#### *3.1. PS, and ZP Analysis*

The average PS and ZP of 15 formulations (CNP1–CNP15) were determined with zeta sizer (PSS NICOMP Z3000, Port Richey, FL, USA). Initially, 1 mL of samples was diluted ten times with double distilled water and mixed gently by handshaking to obtain an appropriate scattering intensity; it was measured at a fixed scattering angle of 90◦. All measurements were taken in triplicate [21].

### *3.2. Determination of DEE (Drug Entrapment Efficiency) and Loading Capacity (LC)*

For determining the DEE, 10 mg of nanoparticles (NPs) was added to 10 mL of acetone to isolate APG into acetone. The samples in acetone were gently ultra-centrifuged at 20,000 rpm for 1 h at 4 ◦C to collect the supernatant. Then, 10 μL of suspension was diluted to 1 mL with acetone and underwent the process of estimations. The appropriate APG concentrations were obtained with a UV-VIS spectrophotometer (Jenway 6850 double beam spectrophotometer, Cole-Parmer, Saint Neots, UK) [4,22]. The following formula was used to calculate the DEE and LC.

$$\text{DEE (\%)} = \frac{\text{Total amount of APG added} - \text{Total amount of APG in compartment}}{\text{Total amount of APG added}} \times 100$$

$$\text{LCC} \left( \% \right) = \frac{\text{Total amount of APG added} \ - \text{Total amount of APG in compartment}}{\text{weight of NPs}} \times 100$$

#### *3.3. Transmission Electron Microscope (TEM) Analysis*

The sample for TEM was prepared using phosphotungstic acid staining. The TEM sample was developed by placing a drop of the NPs suspension on a carbon-coated copper grid. The sample was dried and examined with TEM (JEOL JEM1010, Tokyo, Japan).

#### *3.4. In Vitro Drug Release*

The release study of encapsulated APG from PEGylated-CNPs was studied at two different pH conditions using the dialysis bag diffusion technique, both at 37 ◦C under sink conditions. The selected dialysis bag (M.W 12 kDa, Sigma–Aldrich, St. Louis, MO, USA) was washed and prepared for study according to the manufacturer's guidelines. A release study was carried out in PBS at pH 6.8 and pH 7.4 to estimate the drug's release in tumor surroundings that became slightly acidic and physiological pH, respectively [8,23]. In both studies, samples containing 10 mg APG were installed in dialysis bags and submerged in a beaker of 100 mL PBS with 1% *w/v* Tween 20 retained at 37 ◦C and stirring at 200 rpm. The

sample's aliquots were withdrawn at predefined time points and examined using a UV-VIS spectrophotometer at 335 nm to estimate the release of APG.

#### *3.5. Storage and pH Stability Studies*

Physical and chemical stabilities of the optimized formulation were evaluated at two different temperatures 4 ± 2 ◦C and 25 ± 5 ◦C for periods of 4 weeks to analyze any changes with respect to PS, PDI, and ZP. The formulation's stability was also tested at two different pH levels of 6.8 and 7.4.

#### *3.6. Determination of Antioxidant Potential of Optimized APG-Loaded PEGylated-CNPs*

The antioxidant potential of plant origin materials is defined as their capacity to catalyze the degradation of free radicals, i.e., 1,1-Diphenyl-2-picrylhydrazyl (DPPH), and the ability to significantly reduce their concentration. According to previously published reports, the study was carried out on optimized APG-loaded PEGylated-CNPs and APG suspension to assess their antioxidant potential [24]. The stock solution (10 mg/mL) was diluted with ethanol up until a concentration of 25–250 g/mL. The collected samples (500 L) were added to DPPH solution (0.02 percent in ethanol). The prepared samples were stirred and kept at 25 ◦C in a dark place for 1 h to carry the complete reaction process. At the end of the reaction, the violet color of DPPH changed to being colorless. The same experiment was repeated with blank PEGylated-CNPs. The samples were spectrophotometrically examined at 517 nm. The percentage of antioxidant activity was calculated as follows.

$$\text{Antioxidant activity} \left( \% \text{AA} \right) = \frac{\text{Absorbance of control} - \text{Absorbance of test}}{\text{Absorbance of control}} \times 100$$

#### *3.7. Cytotoxicity Study*

The impact of various concentrations of pure APG and APG-loaded PEGylated-CNPs on MCF7 cells was analyzed along with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). The cytotoxic evaluation via the MTT assay is based on the principle of colorimetric tests, which relies on viable cells' ability to selectively minimize the tetrazolium constituent of MTT to purple-colored formazan crystals [24]. In brief, MCF7 cells were grown into 96-well plates at around 15,000 cells/well density in 100 μL cell media (DMEM, 10% FBS). Seeded cells were kept overnight at 37 °C and 5% CO2 to enable improved adherence. Various concentrations of pure APG and the formulation were placed in different wells to examine their cytotoxic effects. DMSO was also used to produce stock solutions of the standard and the prepared formulation, which were sequentially diluted in a 96-well plate using serum-free media. The final DMSO concentration was kept below 1% to avoid any unwanted effects on the cells. The same diluted DMSO in the cell culture media was considered as the vehicle control. Following several pilot experiments, a range of concentration was chosen for pure APG and optimized formulations, and the assay was conducted in quadruplicate. After 24 h of exposure to APG, each well, except the blank, was filled with 10 μL MTT solution (5 mg/mL PBS). The treated cells were kept for incubation of 4 h to allow the metabolic process of MTT by viable cells. Cell media was removed from each well, and 100 μL DMSO was incorporated to liquefy the formazan of MTT. After the incubation of 30 min, the plate was read at 570 nm using DMSO as blank. The cellular viability was calculated as the viable cell's percentage compared with the control group [25]. The dose–effect curve was also used to calculate IC50 values, which were then expressed as concentrations (μM).

#### *3.8. Statistical Analysis*

Data are displayed as Mean ± SD. Raw data were evaluated with Graph Pad InStat demo version (GraphPad Software Inc., La Jolla, CA, USA). Data were examined using one-way ANOVA along with Tukey–Kramer multiple comparison tests to evaluate the statistical significance at different concentration exposures and the control.

#### **4. Results and Discussion**

In this study, we optimized and developed APG-loaded PEGylated-CNPs for improving therapeutic potential and for investigating the current use of APG in the treatment of cancer. The use of polymeric NPs as a drug carrier was motivated by the fact that it holds unique potential in cancer treatments. This is due to its biocompatibility and safeness with respect to normal tissues, which are attained by hiding the drug's toxicity. An additional significant benefit of NPs is that they improve drug efficacy at the targeted site because of its permeable properties [8]. Furthermore, CS was chosen as a carrier system for NPs because of its inherent positive charge, as well as other advantages including its biodegradability, biocompatibility, and lower cost. The positively charged surface of NPs targets tumor cells to a greater extent than the negatively charged surface of NPs [26]. They are more readily absorbed by cells, and this may damage cell membranes [27]. The mucoadhesiveness of CS relative to the bio-membrane is due to its adhesive nature. Furthermore, cells preferentially consume positively charged CNPs over negatively or neutrally charged CNPs [28]. The key mechanism is electrostatic interaction between the CS's NH3+ groups and negatively charged DNA's phosphoryl groups. The positive surface charge allows binding with negatively charged cell membrane and increases permeation.

To confirm the hypothesis, we used a single-step ionotropic gelation method to make CNPs loaded with APG. CNPs were designed by ionic gelation, which requires two aqueous phases mixture, one of which is the polymer CS and another is a polyanion TPP. The ratio of CS to TPP was optimized to be 1:1. The amino group of CS interacts with the TPP to form a nano-sized complex [29]. Furthermore, CNPs were also coated with PEG400 on the surface to enhance their safety and stability. The surface coatings of CNPs with PEG are based on physical cross-linking using electrostatic interactions. The method used here is a simple and gentle method for the preparation of the formulation. It is preferable to use physical crosslinking rather than chemical crosslinking to prevent excipient toxicity and other undesirable side effects. In particular, this method tries to avoid surfactants and organic solvents, resulting in the preparation of a novel formulation that is both safer and more cost-effective.

Preliminary experiments were carried out to ascertain the composition of PEGylated-CNPs. On the basis of preliminary research, three factors were chosen at three different levels: the amount of CS: TPP (X1), the amount of PEG 400 (X2), and the sonication time (X3). According to selected range of the components, BBD resulted in 15 different APG-loaded PEGylated-CNP formulations, and these were examined with selected response variables, i.e., PS (Y1), % EE (Y2), and ZP (Y3), as seen in Table 2.

#### *4.1. Designed Experiment Statistical Analysis*

The surface response methodology along with BBD is an efficient appliance for the examination of independent variables at a different level with limited experimental runs [20]. In this present study, three-factor three-level factorial designs with respect to responses were investigated. The generated BBD was then used to conduct a series of experiments. The result of measured responses for all formulations are reported in Table 2. The contributing factors and its responses were integrated, with statistical evaluation providing the polynomial equation for evaluating the impact of excipient on responses Y1–Y3. All dependent variables were analyzed with the quadratic model, and 3D surface plots for responses were created. The quadratic model's statistical study found significant *p*-values, indicating a better fit for selected responses (Table 4) [30].

#### **Table 4.** Statistical ANOVA results of the quadratic model.



**Table 4.** *Cont.*

#### *4.2. Evaluation of Response Surface with Polynomial Equation*

The three-dimensional response surface plots shown in Figure 1A–C are helpful for demonstrating the relationship of independent variables with dependent variables. These plots represent the effect of two independent factors on a response at a particular point. Whenever these 3D-plots are critically examined, the characteristic effects of independent variables on each response were observed [16].

**Figure 1.** Three-dimensional response surface plot showing the effect of independent variables (**A**) X1 and X2; (**B**) X1 and X3; and (**C**) X2 and X3 on PS.
