*4.8. In Vitro Dissolution Studies*

To estimate the in vivo release profile of APG, in vitro dissolution analyses were conducted in PBS at pH 6.8 and 7.4. In comparison to free drug suspensions, PEGylated CNPs showed controlled drug released, as observed in Figure 6A,B. A biphasic release was also found with an initial burst release of APG in the early phase, followed by a

delayed release of APG in the later phase (up to 24 h). The optimized CNPs showed a sustained release, but the rate was a little higher in pH 6.8 than in PBS pH 7.4. In 8 h of study, about 74% of the drug was released at pH 6.8 and about 62% was released at pH 7.4. The burst effect could have been caused by APG elution near the surface during nanoparticle preparation, which then quickly dispersed when nanoparticles interacted with the dissolution medium. Because of the swelling or deterioration of the polymer, APG was slowly released later on. The remaining APG in nanoparticles did not entirely release until particles have been completely eroded or dissolved in the dissolution media because of the interaction between both the residual APG and the few amine groups of chitosan.

**Figure 5.** (**A**) Particle size and (**B**) TEM micrograph of optimized APG-PEGylated CNPs formulations.

**Figure 6.** In vitro drug release profile of the optimized APG-PEGylated CNPs in (**A**) pH 6.8 and (**B**) pH 7.4 phosphate buffer.

#### *4.9. Storage and pH-Dependent Stability Studies*

Colloidal systems require a high level of stability. Physical instability is frequently a big obstacle to their clinical application. Optimized PEGylated CNPs were stored for three months at temperatures of 5 and 25 ◦C to assure physical stability. The estimation of stability was carried out based on PS, PDI, and ZP (Table 6). The measured features of prepared formulation were stable and had a statistically insignificant difference, indicating that CNP formulations remained stable during these time periods.


**Table 6.** Stability data.

When the formulation comes in direct contact with different pH conditions, it may experience physical changes that cause particle aggregation. As a result, the formulation's physical stability was tested at various pH levels to which it could come into contact. The stability characteristics of CNPs in various experiments is shown in Table 6. The PS, PDI, and zeta potential were all insignificantly different, ensuring the stability of stored formulations under changing pH conditions [32].

### *4.10. In Vitro Antioxidant Activity: DPPH Assay*

The biological activity of APG is influenced by antioxidant activities. The DPPH assay was applied to measure the antioxidant activity of the APG-PEGylated CNPs formulations in vitro, which relies on the lowering of the DPPH radical in the existence of an antioxidant molecule. DPPH is an incredibly sensitive and commonly used antioxidant assay that is used to evaluate antioxidant activities of a wide range of drugs and drug-delivery systems [16,33]. The test results are reported in Table 7, and it is undeniable that APG has anti-oxidant properties. Because of the presence of CS, weak antioxidant activities were also identified in the blank formulations (13.8 ± 3.1%). The CS alone showed some scavenging activities in a previous study by Anraku M. et al. [33]. These findings also show that CS has antioxidant properties and point to potential pharmaceutical applications. These findings indicate that the APG-PEGylated-CNPs formulation had higher radical-scavenging actions than the pure APG suspension. Overall, these findings show that APG-loaded PEGyalted-CNPs are more effective at scavenging oxygen- and nitrogen-centered radicals and have higher antioxidant capacities.

**Table 7.** In vitro antioxidant activity (% AA) of PEG-CNPs formulation in the presence or absence of APG in comparison with a methanolic solution of the APG and calculated as the inhibition percentage of DPPH radical. Data are expressed as mean ± SD (*n* = 3).


#### *4.11. Cytotoxicity Study*

The possible features of the cytotoxic activity study in MCF-7 cells are depicted in Figure 7. When compared to pure APG, PEGylated CNPs composite exhibited significantly greater concentrations and time-dependent cytotoxicity at 24 h of study. Data reveal that the lower concentrations of both APG standard and APG-PEGylated CNPs formulation have MCF7 cell viability, which decreases as the concentration increases (Figure 7). When considering the effects, it seems that the lower concentrations of the APG formulation had improved growth inhibitory effects in MCF7 cells than the standard APG. These influences and comparisons are depicted in Figure 7. After 24 h of treatment against MCF-7 cells, the IC50 values of PEGylated CNPs and pure APG were observed to be 162 ± 14.54 μM and 1834.1 ± 55.74 μM, respectively. When compared to standard APG, data from the cell viability assay revealed that the PEGylated-CNPs formulation containing APG significantly enhanced these impacts in terms of lowering the IC50. The APG inhibits the growth of MCF7 cells in a concentration-dependent manner, as reported by cell viability assays or MTT assays. However, that could be time-dependent, and the current study did not require a time-dependent cell viability assay.

**Figure 7.** Effect of different concentrations of apigenin standard and apigenin formulation (optimized APG-PEGylated CNPs) on cell viability of A549 cells evaluated by MTT assay. Results are expressed as percentage mean ± SD (*n* = 3) (ns = not significant when compared with control; \* = *p* < 0.05 when compared with control; \*\*\* = *p* < 0.001 when compared with control; NS = not significant when compared with same concentration group of apigenin standard; ### = *p* < 0.001 when compared with the same concentration groups of apigenin standard).

This can be explained by the fact that positively charged surface NPs have a higher chance of attacking tumor cells and are more easily gained by cells, potentially causing cell membrane destruction [29,30]. The important mechanism is the electrostatic interaction of the positively charged NH3+ groups of CS with the negative charge entity of DNA. They seem to have a total positive surface with respect to NPs, which allows them to bind with plasma membrane and increases the permeation through it [8].

#### **5. Conclusions**

In the present inspection, BBD was successfully used to design and formulate PE-Gylated APG-encapsulated CNPs. BBD had been used to generate design spaces, and three independent factors were used to achieve an optimized formulation that resulted in a smaller size, optimal ZP, and a higher % DEE. The single-step ionotropic gelation technique was used to make CNPs. PEG 400 was also applied to the surface of the NPs to ensure their safety and stability. For APG-PEGylated CNPs, the optimized formula yielded PS, ZP, and % DEE of 139.63 ± 5.67 nm, 24.68 ± 1.84, and 79.55 ± 3.12 respectively. TEM micrographs revealed a smooth surface and a spherical shape. According to the

release study, PEGylated CNPs had a sustained release pattern for 24 h of periods. Once compared to pure APG, APG-loaded PEGylated CNPs had significantly higher antioxidant capacity. The formulation showed increased dose-dependent antitumor activities in breast cancer cells in comparison to pure APG. As a result, the study's conclusive result is that PEGylated CNPs improved the therapeutic potential and expanded the current use of novel formulations of APG in cancer treatments.

**Author Contributions:** M.A.M.: Conceptualization, investigation, methodology, and writing original draft preparation; N.M.A.: formal analysis and investigation; S.M.A.: methodology and data curation; M.Y.: funding and investigation; M.K.A.: writing—reviewing and editing; M.A.R.: reviewing and editing; A.P.: formal analysis and validation. All authors have read and agreed to the published version of the manuscript.

**Funding:** The current research is supported by Taif University Researchers Supporting Project number (TURSP—2020/293), Taif University, Taif, Saudi Arabia.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The current research is supported by Taif University Researchers Supporting Project number (TURSP—2020/293), Taif University, Taif, Saudi Arabia. The authors also gratefully acknowledge the Northern Border University for providing necessary facilities for the research study.

**Conflicts of Interest:** There are no conflicts of interest reported by the authors.
