Histopathological Analysis

Since the histological study of doxorubicin multi-organ toxicity is a wide topic, we focused on the toxicity of vital organs and a reproductive system that can be harmed by cancer and its associated treatments, and we evaluated the protective activity of our formulation against organ toxicity reported in previous literature [45,58–62]. Liver, kidney, heart, and testis specimens were cut and fixed in a 10% neutral buffered formalin. The formalin-fixed samples were routinely processed, embedded in paraffin, and sectioned. Serial 3 µm sections, serially dehydrated in an ethanol gradient, were stained with Mayer's hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma, St. Louis, MO, USA) and examined microscopically. The histological evaluation was performed by a histopathologist (Dr Mahmoud Soliman) in a blind fashion on coded samples, and a comparison was made with the sections from the control.

#### *4.3. Statistical Analysis*

All experiments were performed in triplicate and results were expressed as means ± standard deviations (SD) except for the in vivo study where the results were expressed as mean ± standard error (SE) for eight animals in each group (n = 8). Statistical analysis tests were performed using SPSS 21.0 software. For all results, the differences were considered significant when *p* < 0.05, and they were regarded as extremely significant when *p* < 0.0001 presented by \* and \*\*\*, respectively. Comparisons between groups were performed using one-way multivariate ANOVA followed by a Tukey's post hoc test or Student t-test where appropriate.

#### **5. Conclusions**

Based on the findings presented in this study, it is possible to conclude that the desolvation method successfully produced SML-ANPs with promising physicochemical features, such as high stability, sustained release and high loading capacity, where the particle size, zeta potential, and loading capacity of the selected formulation were 127.24 nm, −26.21 mV, and 96.89 g/mg ANPs, respectively. In addition to the specific role of albumin molecules in cellular endocytosis, particles with diameters less than 200 nm improve cellular absorption. Albumin nanoparticles also enhanced pharmacokinetic characteristics. Sesamol suffer from low bioavailability, and they displayed a short half-life and a rapid

elimination rate of 1.25 h and 0.53 L/h, respectively. However, albumin can act as an SML reservoir in the bloodstream with an extended half-life and slower elimination rates of 8.9 h and 0.11 L/h, respectively. SML-ANPS exhibited significant protective effects against hepatocytes pretreated with doxorubicin and the DOX-induced acute toxicity in albino rats as an oxidative stress animal model by down regulating the production of harmful free radicals and the LPO signaling pathway in serum and different organ tissues. Cotreatment with SML-ANPs significantly reduced the elevated serum CK and LDH by 59.3 and 64.5%, respectively, while AST and ALT were inhibited by 60.5 and 59% in comparison to the corresponding values of the free SML treated group: 35.6, 47.2, 49.9, and 41.2%, respectively. As a result, sesamol-loaded albumin nanoparticles may be considered a viable and potentially clinically applicable nano-based platform for the treatment of cancer and inflammatory illnesses in the future. However, more research into their medicinal potential is necessary before use in clinical application.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ph15060733/s1, Figure S1: DSC thermograms of lyophilized SML, HAS, SML/HSA physical mixture, SML/HSA mixture after incubation, and SML-ANPs; Figure S2: FTIR spectra of HSA (A) SML (B) SML/HSA physical mixture (C) SML/HSA mixture after incubation (D), and SML-ANPS (E) together with the successful docking of sesamol on HSA (F); Table S1: Physicochemical characteristics of SML-ANPs; Table S2: The effect of the factors controlling SML-ANPs preparation and their levels in the D-optimal design, where pH is the most significant factor; Table S3: Release kinetics of SML-ANPs (F14); Table S4: Hepatoprotective effect of free SML and SML-ANPs on rat hepatocytes measured by the MTS assay; Table S5: The histopathological scores of the liver, kidney, heart, and testis in each groups <sup>a</sup> ; Figure S3: Dose-response curve for sesamol effects on cell viability of rat hepatocytes to calculate the CC 50. The values are presented as mean ± SD (n in each group = 3); Table S6: Evaluation of sesamol cytotoxicity against Rat hepatocytes cell line after 48 h [50,63–65].

**Author Contributions:** Conceptualization, R.M.H.; data curation, S.Z., M.E.S., M.E. and R.M.H.; formal analysis, M.E.S. and R.M.H.; investigation, M.E.S. and M.E.; methodology, S.Z., M.E.S. and R.M.H.; project administration, R.M.H.; software, S.Z. and R.M.H.; supervision, M.E.S., M.E. and R.M.H.; validation, S.Z.; visualization, M.E.S., M.E. and R.M.H.; writing—original draft, S.Z.; writing review and editing, M.E.S., M.E. and R.M.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The animal study protocol was approved by the animal care and use committee of the faculty of Pharmacy, Ain shams University, Cairo, Egypt (no.155, 2017).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article and supplementary materials.

**Acknowledgments:** The authors thank Hossam El-Din M Omar, Department of Zoology and Dean of Faculty of Science, Assiut University, Assiut, Egypt. The biochemical assessment and the preparation of histology slides were facilitated by the laboratory of the physiology of Zoology department, Faculty of Science, Assiut University under his supervision. The authors also would like to thank Mahmoud Soliman, department of Pathology and Clinical Pathology, Faculty of Veterinary Medicine, Assiut University, for the professional grading of the histology slides.

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

#### **Abbreviations**

ALT: Serum alanine aminotransferase; ANOVA: Analysis of variance; ANPs: albuminbased nanoparticles; AST: Aspartate transaminase; AUC0–∞: The area under the curve; CK: Creatine kinase; Cmax: The maximum plasma concentration; CV%: Coefficient of variation; DAD: Diode-Array Detector; DL%: Percent of the drug loading; DOE: Design of experiments; DOD: D-optimal design; DOX: Doxorubicin; DLS: dynamic light scattering; DSC: Differential scanning calorimetry; EE%: Entrapment efficiency; Fe2+: iron; FT-IR: Fourier transform-infrared spectroscopy; GA: glutaraldehyde; GP: Group; HO•: hydroxyl radical; HSA: Human serum albumin; IV: intravenous injection; LDH: Lactate dehydrogenase; LoQ: Limit of quantification; LPO: Lipid peroxidation; MDA: Malondialdehyde; MRT0–∞: Mean residence time; MTT: 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; USA: The United States of America; PBS: Phosphate buffered saline solution; PDI: Polydispersity index; PS: Particle size; ROS; reactive oxygen species; SD: Standard deviations; SE: Standard error; SEM: Scanning electron microscopes; SML: sesamol; SML-ANPS: Sesamol-loaded albumin nanoparticles; SPSS: Statistical Package for the Social Sciences; TBA: Thiobarbituric acid; TCA: Trichloroacetic acid; T1/2: the half-life; Tmax: the time of maximal concentration; %Y: Yield; ZP: Zeta potential.
