4.6.7. Proton-Nucleic Magnetic Resonance (1H-NMR)

The <sup>1</sup>H-NMR spectra of Dox–Erlo NPs and Dox–Erlo-NP conjugates were acquired on a Bruker Avance—II (Terre Haute, IN, USA) at 400 MHz. The chemical shifting was reported in ppm for the structure elucidation of Dox–Erlo-NP conjugates, which were compared with the Dox–Erlo NPs to confirm the conjugation by using a DMSO solvent and investigating the surface chemistry of the nano-conjugate [56].

#### 4.6.8. In Vitro Release Studies

In vitro Dox and Erlo release of the developed formulations of Dox–Erlo NPs and Dox– Erlo-NP conjugates were assessed by diluting the nanoparticles in PBS at a physiological pH of 7.4 and at an acidic, intracellular, endosomal pH of 5.4. The encapsulated drug NPs were kept enclosed in a dialysis bag (Mol. wt cut-off = 60–8 kDa) with the ends tightened. The dialysis bag was then immersed in 50 mL of PBS at a pH of 7.4 and maintained at 37 ± 0.5 ◦C with a gentle shaking at 50 rpm. A sample (1 mL) was adjourned at programmed intervals and replaced with an equal volume of fresh PBS at a pH of 7.4 and a pH of 5.4. Samples were examined using UV-visible light with wavelengths of 342 nm and 480 nm.

#### 4.6.9. Hemolysis Study

The hemolysis study was carried out by collecting blood from adult rats in EDTAcoated tubes, followed by centrifugation at 2000 rpm for 10 min to separate the cells and plasma. Further, the sediment cells were washed (*n* = 3) with PBS at a pH of 7.4. Various concentrations of NPs (placebo NPs, Dox–Erlo NPs, and Dox–Erlo-NP conjugates), including 1.5 mg, 3 mg, and 6 mg, were incubated with RBCs of number 1.5 <sup>×</sup> <sup>10</sup><sup>7</sup> at 37 ◦<sup>C</sup> for 1 h. The samples were subsequently subjected to centrifugation for ten minutes at 2000 rpm. The supernatant was analyzed at 540 nm using UV-visible spectroscopy. An RBC hemolysis of 100% with Triton X-100 was considered to be a positive control and an RBC hemolysis of 0% after treatment with PBS was considered to be a negative

control [57]. If the hemolysis was less than 10%, it was regarded as non-toxic. The following formula was used to calculate the hemolysis %:

$$\% \text{ Henrylysis} = [(\text{Abs}(\text{treatment}) - \text{Abs}(\text{PBS}))/\text{Abs}(\text{Tortion} \times -100)] \times 100 \tag{2}$$

## 4.6.10. Cytotoxicity Study

The test for the efficacy of the nanosystem composition in terms of therapeutic accumulation and internalization was investigated in cell lines C6 (ATCC, CCL107) and U87 (ATCC, HTB14) of glioma tumors in vitro. The cell-viability study of Dox–Erlo NP and Dox–Erlo-NP conjugate nanosystem was carried out using an MTT assay. The culture cells were treated with DMSO, and the formazan reagent formed (solubilized) was estimated using a spectrophotometer. MTT acts only on biologically active cells, and the activity of cells indicates the cells' viability [58]. In this method, cell lines were added to a 96-well plate (106 cells/well) containing DMEM media and then incubated overnight at 37 ◦C in a humidified atmosphere in which the air was enriched with (5% *v/v*) CO<sup>2</sup> tofacilitate the attachment of cells to the bottom of each well. Upon the well attachment of cultured cells, cells were treated with concentrations (between 0.20 and 6.4 µM) of Dox–Erlo-NPs and Dox–Erlo-NP conjugates and incubated for 24 h.

After the completion of the treatment, the media were removed carefully and incubation was repeated with 10 mL of MTT for 3 h. After the completion of the incubation, the optical density was measured at 570 nm in a microplate reader. Each experiment was performed in triplicate (*n* = 3). Untreated cells were related to the control group (100% cell viability), and the IC50 of the cells was determined. IC50 is the drug concentration that slows cell growth by 50% when compared to a control. It is calculated using a regression analysis of cell-viability studies.

Cell viability (%) was expressed as the mean viability (%) ± standard deviation (SD) (*n* = 3) using the following formula:

The cell viability (%) was represented as mean ± SD (*n* = 3) using following formula;

$$\text{Percent cell viability} = \text{OD treated} / \text{OD controlled} \times 100 \tag{3}$$

#### 4.6.11. Biodistribution Studies

Animals were procured from an animal house prior to the experiment and maintained in polymeric cage as per animal ethical guidelines. The animals were housed at room temperature and exposed to 12 h of light/dark. They were kept on food and water ad libitum. Institutional Animal Ethics Committee (IAEC) guidelines were followed in conducting animal experiments as per the guidelines by DIT University, Dehradun, Uttarakhand, India (Ref no. DITU/IAEC/21-22/07-05). To investigate biodistribution, the administration of a single dose of a formulation such as pure Erlo, pure Dox, Dox–Erlo NPs, or Dox–Erlo-NPs conjugates having 1 mg Erlo and 5 µg of Dox was performed via the nose-to-brain delivery of 20 µL once per day for 14 days in four groups of male Wistar rats (*n* = 3). Different organs, viz., the liver, heart, kidney, blood, and brain were removed from each group (*n* = 3) 24 h after the last dose. The removed tissues were blotted with tissue paper, weighed, and homogenized in 1 mL of ice-cold sodium chloride solution per 1 g of tissue. Thereafter, aliquots were separated and kept at −20 ◦C until analysis. The Dox and Erlo contents were estimated by using HPLC, using the procedure shown in the section on HPLC methodology.

#### 4.6.12. Stability Study

This study was carried out as per ICH guidelines on three months of Dox–Erlo-NP conjugates. A stability study of the in-house-built formulation was performed to ensure the physiochemical alteration in the quality of Dox–Erlo-NP conjugates. The samples were kept in a stability chamber at an ambient temperature, 25 ± 2 ◦C, 65 ± 5% RH; and at a higher temperature, 40 ± 2 ◦C, 75 ± 5% RH, for 90 days. The evaluations were conducted at intervals of 0, 30, 60, and 90 days.

#### 4.6.13. Statistical Analysis

Quantitative data are presented as the mean ± standard deviation (SD). Statistical comparisons between different treatments were analyzed with a one-way ANOVA using Graph Pad Prism. A value of *p* < 0.05 was considered statistically significant.

#### **5. Conclusions**

Dox–Erlo NPs were successfully developed for the first time, prepared using a *Cinnamomum zeylanicum* biopolymer. The optimization procedure was accomplished using a three-factor and three-level Box–Behnken experimental design. The optimized composition had a biopolymer content of 2.94% *w*/*v*, a surfactant content of a 2.2% *w*/*v*, and a sonication time of 11.39 min. In this optimum composition, formulation was characterized by a particle size of 95.35 nm, a PDI of 0.102, and % drug release of 89.91%. The analytical findings confirmed that both drugs were loaded in the biopolymeric core of the NPs. The biodistribution study revealed that folate-functionalized NP conjugates showed improved Dox and Erlo transport across biological barriers and potentially enriched the drug concentration in the brain. The higher cell death in an MTT assay recorded for the NP conjugates over the drug suspension against C6 and U87 cell lines resulted in an enhanced anti-tumor efficacy. The hemolysis study demonstrated that Dox–Erlo-NP conjugates were suitable for in vivo administration. Based on the findings of the studies, it is further suggested that Dox–Erlo-NP conjugates could be an option for effective drug delivery to glioma cancer.

#### **6. Patents**

This work has been patented as Indian Patent Application No. 202111038933. Publication Date, 10 September 2021.

**Author Contributions:** Conceptualization, M.H.A. and M.F.; methodology, M.H.A. and M.F.; software, M.H.A. and M.F.; validation, M.H.A. and M.F.; formal analysis, M.H.A., M.F. and H.C.; investigation, M.H.A. and H.C.; resources, M.J., A.-H.E. and M.S.A.; data curation, M.F., M.H.A. and H.C.; writing—original draft preparation, M.F.; writing—review and editing, M.H.A., H.C., M.J. and A.-H.E.; visualization, M.S.A., M.J., F.T. and A.-H.E.; supervision, M.H.A. and H.C.; project administration, M.H.A. and H.C.; funding acquisition, M.S.A., M.J., F.T. and A.-H.E. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Dean of Scientific Research, King Khalid University, Saudi Arabia [grant numbers: RGP.1/242/43].

**Institutional Review Board Statement:** The animal study protocol was approved by the Institutional Animal Ethics Committee (IAEC) guidelines of Faculty of Pharmacy, DIT University (Ref no. DITU/IAEC/21-22/07-05, approved on 1 July 2021) for studies involving animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** This work was supported by the Faculty of Pharmacy, DIT University, Dehradun. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through the Small Groups Project under grant number (RGP.1/242/43). M.J. and Abdel thanks to King Abdullah University of Science and Technology, Saudi Arabia for financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.
