*3.1. Preparation and Optimization of β-CD-NS*

NS formulations were prepared using DPC as a cross-linker for β-CD hydroxyl groups with the formation of new carbonate bonds [30]. A molar ratio 1:6 of β-CD:DPC was selected based on previous reports showing a higher percentage yield compared to other ratios tested [10,30]. Optimization of the cross-linking reaction conditions was carried out by changing the temperature, time, and volume of the reaction vehicle (DMF) (Table 2). The successful formation of NS was primarily confirmed by the observation of a gel-like mass during the reaction (gelification) together with the appearance of a deep violet color upon adding ferric chloride [30]. As shown in Table 2, using a small volume of DMF (3 mL) resulted in an incomplete reaction at different reaction conditions (F1–F4). Increasing the DMF volume to 6 mL resulted in the successful formation of NS, except for F5 (90 ◦C, 2 h). Furthermore, increasing the reaction temperature to 120 and 150 ◦C allowed for NS formation in the shorter time interval tested (2 h, F7, and F9). Based on the percentage yield (49.85 ± 1.2%), 150 ◦C was selected as the optimum reaction temperature (F9).


**Table 2.** Optimization of cross-linking reaction conditions.

FS loading (FS-NS) was achieved by simple overnight stirring of drug ethanolic solution and the aqueous nano-dispersion of blank NS (at a weight ratio of 1:4) [10].

### *3.2. Preparation of LF-Coated FS-NS*

LF has been extensively studied as a natural tumor-targeting ligand owing to its ability to bind specific receptors overexpressed on the surface of cancerous cells, in addition to its biocompatibility and biodegradability [29]. In the current work, LF coating on NS was investigated for the first time. This was based on the electrostatic interaction between cationic LF and the negatively charged surface of FS-NS (−26 mV). The effect of the LF concentration added (25–100 mg/mL) on the ζ-potential of FS-NS was studied (Figure 1). Upon increasing the LF concentration from 0 to 75 mg/mL, ζ-potential shifted from −26 ± 6.5 to 24 ± 1.1 mV, indicating the deposition of LF on the surface of NS. Further increases in LF beyond 75 mg/mL only brought about a slight insignificant (*p* ≥ 0.05) change in ζ-potential (25.1 ± 2 mV for 100 mg/mL LF), reflecting complete FS-NS surface coverage with an LF layer. Hence, 75 mg/mL was chosen as the optimum LF concentration for coating FS-NS.

**Figure 1.** Effect of lactoferrin concentration on FS-NS ζ-potential (mV). Data presented as mean ± SD (n = 3).

### *3.3. Physicochemical Characterization*

### 3.3.1. Colloidal Properties and Entrapment Efficiency

The colloidal properties of selected NS formulations are shown in Table 3. The optimized blank NS formulation (F9) chosen for further development showed an average PS, PDI, and ζ-potential of 46.1 ± 6.2 nm, 0.13, and −22 ± 0.8 mV, respectively. The negative charge could be attributed to the presence of free β-Cyclodextrin hydroxyl groups and DPC carbonyl groups [10,41]. FS loading into NS resulted in a slight decrease in PS (38.2 ± 3.8 nm) with an insignificant (*p* ≥ 0.05) change in ζ-potential (−26 ± 6.5 mV). These results reflect efficient drug entrapment in the porous NS. LF-coated FS-NS showed a significant increase in PS (*p* ≤ 0.05) compared to the uncoated formulation. The increase in size, together with the shift in ζ-potential from negative to positive, suggests successful coating and deposition of LF on the surface of NS [26]. The high ζ-potential value of the three NS formulations tested allows for sufficient particle repulsion, indicating good colloidal stability [41].


**Table 3.** Physicochemical properties of the selected nanosponge formulations (n = 3).

A high entrapment efficiency of FS (>95%) that was unaffected by LF coating, and DL% ~24%, were calculated. (Table 2). High EE% of lipophilic drugs in ß-CD nanosponge formulations have been previously reported for curcumin [42] and quercetin [10]. The high EE% could be ascribed to the porous nature of NS, in which the drug is deposited [43].

### 3.3.2. Analysis of Surface Area and Porosity of NS

Figure 2 illustrates the nitrogen adsorption–desorption isotherm (Figure 2A) and the pore size distribution (Figure 2B) of the selected NS formulation (F9). A type IV isotherm with a hysteresis loop and an average pore diameter of ~26 nm were obtained, reflecting the mesoporous nature of NS [44]. The total surface area and total pore volume were 15.8 m2/g and 0.1 cm3/g, respectively. The large surface area and pore volume observed allow for efficient drug loading [31], subsequently supporting the high FS EE% observed.

### 3.3.3. Microscopical Examination

SEM imaging showed a rough, porous surface (Figure 3a) with a pore diameter of ~30 nm, confirming the mesoporous structure of NS. TEM micrographs revealed spherical non-aggregated nanoparticles for blank NS (Figure 3b), which was not affected by FS loading except for the decrease in particle size (Figure 3c). A dense coat surrounding the nanosponges could be observed for LF-FS-NS (Figure 3d).

**Figure 3.** (**a**) SEM of NS with measurement of pore size. Magnification × 40 K, scale bar represents 500 nm. TEM images (**b**–**d**) showing the morphology of (**b**) NS, (**c**) FS-NS, and (**d**) LF-FS-NS. Magnification × 30 K, scale bar represents 100 nm.

### 3.3.4. In Vitro Drug Release

In vitro release profiles of FS solution, FS-NS, and LF-FS-NS are shown in Figure 4. FS solution exhibited complete release, reaching 100% after 3 h. On the other hand, FS release from FS-NS and LF-FS-NS was slow and steady, reaching only 35 and 26% after 24 h, respectively. In addition to the prolonged sustained release profile, the low initial burst observed (less than 10%) strongly confirms the presence of FS inside the pores of the nanosponge, as previously stated for other drugs loaded in ß-CD-NS [12,13]. The decrease in the slope of the drug release curve with time reflects the gradual increment of diffusion distance in the polymeric matrix. Moreover, the lower percentage of FS released for LF-FS-NS as compared to FS-NS could be attributed to the additional barrier created by the coating layer restricting release medium diffusion into the NS matrix [45].

**Figure 4.** In vitro release profile of FS from FS solution, FS-NS, and LF-FS-NS at 37 ◦C in PBS (7.4) with 0.1% *<sup>w</sup>*/*<sup>v</sup>* Tween® 80. Data represent mean <sup>±</sup> SD (n = 3).

The drug release mechanism of FS-NS and LF-FS-NS was determined by fitting to different release kinetics models, namely the zero-order, first-order, Higuchi, Korsmeyer –Peppas, and Hixson–Crowell models. To designate the data best fit, the largest correlation coefficient (r) and smallest mean standard error (MSE) were used as statistical parameters. The results indicated diffusion-controlled FS release from both FS-NS and LF-FS-NS as the greatest r value (0.94), and the minimum MSE value were observed for the Korsmeyer– Peppas model. A release exponent value (n) ≤ 0.5 was observed as being indicative of Fickian diffusion.

### 3.3.5. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra of β-CD, DPC, FS, blank NS, and FS-NS are shown in Figure 5A. β-CD displayed characteristic bands at 3288 for O–H stretching, 2921 for C-H stretching, 1416 for C-H bending, and 1047 cm−<sup>1</sup> for C–O stretching. The most characteristic band in the FTIR spectrum of DPC is 1771 cm−1, corresponding to C=O. In the NS spectrum, the appearance of a new sharp absorption band at 1759 cm−<sup>1</sup> corresponding to carbonyl (C=O) stretching vibration, which was absent in the FTIR spectrum of pure β-CD, together with the reduction in O–H stretching vibration, indicates carbonate linkage formation with OH groups of β-CD, proving efficient cross-linking with DPC. This is consistent with previous reports [10,12].

The FS spectrum showed absorption bands at 3519 and 3351 cm−<sup>1</sup> corresponding to aromatic ring attached O–H stretching, 1607 cm−<sup>1</sup> for C=O stretching, 1571 cm−<sup>1</sup> for C=C stretching, 1477 cm−<sup>1</sup> for C–O stretching, and 1279 cm−<sup>1</sup> for C–O–H bending vibrations [26].

The FTIR spectrum of FS-NS exhibited some changes in the fingerprint region (400–1400 cm−1) compared to the FS spectrum. Peak broadening and shifting in the spectrum of FS-NS compared to blank NS suggest definite interactions between FS and β-CD NS, further confirming the effective internalization of the drug into the pores of the formed NS [10].

**Figure 5.** (**A**) FTIR spectra for FS-NS and its components; (**B**) X-ray diffraction pattern of FS, NS, and FS-NS.

### 3.3.6. X-ray Diffractometry (XRD)

To assess drug crystallinity in the developed FS-NS, nondestructive XRD was conducted. Figure 5B shows the XRD patterns of FS, NS, and FS-NS, as well as Aerosil® 200, which was used for drying the aqueous dispersions of tested NS. The Aerosil® 200 diffractogram showed its amorphous structure, as only a broad peak at 22.5◦ was observed [36]. The FS diffractogram revealed prominent sharp diffraction peaks at 2θ angles 12.5, 15.2, 17.1, 25.7, and 28.5◦, reflecting FS's crystalline nature [46]. These characteristic sharp peaks of FS disappeared upon loading into FS-NS, indicating its presence in an amorphous state which could be attributed to the supramolecular complex formation of FS with NS [47].

### *3.4. Cell Line Studies*

### 3.4.1. Cytotoxicity Evaluation

The antiproliferative effect of FS on TNBC has been previously reported [37,48,49]. In this study, the cytotoxicity of FS-NS and LF-FS-NS in comparison with FS solution was evaluated on MDA-MB-231 cells using MTT assay in the concentration range of 10–100 μg/mL. To compare the antitumor activity of FS solution and NS formulations, %viability and, consequently, IC50 values were calculated. FS showed concentration-dependent cytotoxicity with an IC50 value of 59.18 μg/mL. Interestingly, FS encapsulation into NS (FS-NS) resulted in a 1.3-fold decrease in IC50 (45.06 μg/mL). The obtained results are in agreement with previous reports on the promotion of cytotoxicity of different biomolecules following loading into ß-CD-NS formulations [12,13]. Further coating of FS-NS with LF boosted the antiproliferative effect of FS with a reduction in IC50 to 27.67 μg/mL (a 2.1- and 1.6-fold decrease compared to FS and FS-NS, respectively). Similar effects of LF modification on cytotoxicity enhancement were shown for PLGA nanoparticles [28], bilosomes [33], and nanostructured lipid carriers (NLC) [50]. This could be partly a result of the innate anticancer effect of LF [29]. Blank formulations (NS and LF-NS) were included in the study and showed percentage MDA-MB-231 cell viability exceeding 80% for all concentrations tested. This proves the cytocompatibility of the blank nano-formulations and provides

evidence that the observed cytotoxicity of FS-loaded nanosponge formulations is due to the enhanced cellular interactions of FS following loading into NS [26].

### 3.4.2. In Vitro Apoptosis Assay

The apoptosis (programmed cell death)-mediated anticancer effect of FS and FSloaded nanosponge formulations was investigated using flow cytometry of annexin Vstained apoptotic cells (Figure 6A). FS resulted in a significant (*p* ≤ 0.05) apoptotic activity compared to the control group (23.9 ± 0.17% and 7.3 ± 0.28 %, respectively). Loading into NS formulation brought about a significant increase in FS apoptotic activity (29.9 ± 0.3%) which was further augmented (*p* ≤ 0.05) following LF coating of FS-NS (36.6 ± 0.61%). The significant apoptotic effect of LF-FS-NS compared to the uncoated formulation could somewhat be attributed to the inhibitory effect of LF on plasmalemmal V-H+-ATPase [51]. The higher cellular apoptosis for FS-NS and LF-FS-NS formulations compared to the FS solution elucidates in part the observed enhancement of cytotoxicity.

**Figure 6.** (**A**) Percentage apoptosis for FS, FS-NS, and LF-FS-NS by flow cytometry using annexin V FITC/propidium iodide assay after incubation for 24 h with MDA-MB-231 cells and (**B**) scratch wound assay: (a) migration inhibitory activity of free FS, FS-NS, and LF-FS-NS on MDA-MB-231 cells (magnification ×20) and (b) percentage wound closure. Data were expressed as means ± SD (n = 3). Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test for group comparisons. Means of similar symbols were statistically insignificant: a>b>c>d(*p* ≤ 0.05).

### 3.4.3. Cell Migration

FS has been previously shown to suppress migration and metastasis of TNBC through epithelial-to-mesenchymal transition reversal via the PTEN/Akt/GSK3β signaling pathway [20]. In the current study, the ability of FS-NS and LF-FS-NS vs. FS solution to

inhibit MDA-MB-321 cell migration was studied by the wound healing assay (Figure 6B). Compared to control cells, FS significantly reduced wound closure by two-fold. This is in agreement with previous reports on the ability of FS to effectively reduce the migration of TNBC cells in the range of 20 to 76% [52]. Interestingly, FS-NS succeeded in further significant (*p* ≤ 0.05) inhibition of wound closure (42.5 ± 3.5 and 30.1 ± 2.1% for FS solution and FS-NS, respectively). LF-FS-NS was the most suppressing formulation, achieving an approximately 15-, 8-, and 6-fold decrease in wound closure compared to the control, FS solution, and FS-NS, respectively.

### 3.4.4. Cellular Uptake

The extent of the internalization of C6-labeled NS and LF-NS by MDA-MB-231 cells was evaluated by confocal microscopy (Figure 7). Following 4 h exposure, the free dye was internalized by the cells; however, C6 loading in NS resulted in a 3- and 6-fold increase in cellular uptake for the uncoated and LF-coated formulations, respectively. The improvement in cellular uptake following FS loading into NS and LF-NS could partially explain the enhanced antiproliferative, apoptotic, and migration-inhibitory effects observed. Enhanced cellular uptake of the nano-formulations could be attributed to their small size (below 100 nm) which allows for endocytosis and not just simple diffusion as previously reported [33]. The superior cellular uptake observed for LF-C6-NS compared to C6-NS reflects faster and more efficient internalization of the formulation into breast cancer cells due to LF interaction with its target receptors overexpressed on metabolically active cancer cells [28]. Moreover, LF's positive charge allows for cell entry via electrostatic interaction with the negatively charged cell membrane glycosaminoglycans [53]. Surface charge was shown to not only affect cellular uptake rates but also intracellular trafficking [50].

**Figure 7.** (**a**) Confocal laser scanning microscope images showing cellular uptake of free coumarin 6 solution and its NS formulations after 4 h incubation with MDA-MB-231 cells and (**b**) corrected total fluorescence intensity. Data were expressed as means ± SD (n = 3). Data were analyzed using one-way ANOVA followed by Tukey's post-hoc test for group comparisons. Means of similar symbols were statistically insignificant: a>b>c(*p* ≤ 0.05).

### *3.5. In Vivo Studies*

### 3.5.1. Pharmacokinetic Study

The bioavailability of FS administered to rats either orally or by IP injection was studied for free FS, FS-NS, and LF-FS-NS (Table 4, Figure 8). The drug plasma concentration– time profiles after oral administration of a single FS dose (30 mg/kg) either free or loaded into NS formulations are demonstrated in Figure 8a, and the calculated pharmacokinetic parameters are listed in Table 4. Results revealed marked changes in the pharmacokinetic behavior of FS after loading into both uncoated and LF-coated NS. A significant decrease in Tmax was observed for LF-FS-NS (0.25 h) compared to FS suspension and FS-NS (1 h) indicating a faster rate of absorption. This might be attributed to the positive charge on LF-FS-NS, which allows for favorable distribution in the small intestine and uptake via multiple endocytosis pathways compared to negatively charged nanoparticles [54]. Furthermore, positively charged nanoparticles were shown to electrokinetically interact with mucus, thus, opening epithelial cells' tight junctions and promoting absorption via the paracellular pathway [55].

**Table 4.** Pharmacokinetic parameters of FS after oral and IP administration of a single dose (30 mg/kg) of FS suspension and NS formulations in rats.


The study was conducted on female Wistar rats with six animals in each group. Values were expressed as mean ± SD. Data were analyzed using one-way (ANOVA) followed by post-hoc test (Tukey's). The level of significance was set at *p* ≤ 0.05. Means of similar symbols are statistically insignificant, a > b > c. \* Relative bioavailability was calculated by dividing AUC 0–<sup>∞</sup> of different NS formulations to that of FS suspension.

Whereas FS loading into uncoated NS did not affect Tmax, a significant increase in Cmax by 1.7-fold (41.3 ± 7.4 ng/mL) compared to FS suspension (24.3 ± 4.6 ng/mL) (*p* ≤ 0.001) was observed. Moreover, LF-FS-NS resulted in a 2.3-fold increase in Cmax (56.2 ± 5.9 ng/mL) compared to FS suspension, which was also significantly higher than the uncoated formulation, (*p* ≤ 0.01). Again, both NS formulations achieved a significant increase in AUC 0–∞, with 3.1- and 2.5-fold for the uncoated and LF-coated NS, respectively, compared to drug suspension (*p* ≤ 0.05), with a significant increase in t1/2 (*p* ≤ 0.05). It is noteworthy that LF-FS-NS exhibited a significantly lower t1/2 compared to FS-NS (*p* ≤ 0.05), owing to its cationic nature, which could facilitate serum protein aggregation, opsonization, and, thus, systemic clearance by macrophages [56]. Despite their difference in t1/2, the differences between AUC for FS-NS and LF-FS-NS did not reach statistical significance.

The pharmacokinetic behavior of FS-NS and LF-FS-NS vs. FS suspension was also investigated following IP administration. Figure 8b shows the drug plasma concentration– time profiles and Table 4 demonstrates the main pharmacokinetic parameters. Following IP administration, an obvious increase in the amount and extent of FS reaching the circulation and, hence, bioavailability, was observed for NS formulations compared to FS suspension, as indicated by the significantly higher Cmax (3-fold for FS-NS and 1.7-fold for LF-FS-NS) and AUC0–<sup>∞</sup> (4.3-fold for FS-NS and 3.2-fold for LF-FS-NS) (*p* ≤ 0.05). Furthermore, FS half-life was significantly prolonged following its loading into either coated or uncoated NS formulations, (*p* ≤ 0.05). Nevertheless, t1/2 for FS-NS was significantly higher than that of LF-FS-NS (*p* ≤ 0.001) in a similar manner to that observed following its oral administration, suggesting a possible systemic clearance by macrophages being positively charged [56].

The improved drug bioavailability following administration of FS-NS formulations via either oral or IP routes compared to FS suspension matches previous reports on the potential of NS to enhance erlotinib bioavailability as a result of supramolecular complex formation between the drug and porous NS [57]. Drug inclusion within the nanocavities reduced its particle size and, accordingly, increased solubility and dissolution rate, hence, facilitating absorption [57]. Furthermore, being deeply incorporated in the nanopores could possibly allow for the avoidance of drug pre-systemic intestinal and first-pass hepatic metabolism [57].

It is worth mentioning that FS bioavailability was significantly higher via the IP route compared to the oral one when administered either as a suspension or loaded into NS formulations. This could be attributed to by-passing the drug pre-systemic intestinal metabolism following IP injection [58]. Furthermore, IP administration allows for a higher and faster drug absorption rate due to the rapid uptake of drug from the peritoneal cavity, resulting in a more rapid saturation of the drug-metabolizing enzymes than following oral administration. Consequently, higher concentrations of the unmetabolized drug will be available in systemic circulation, resulting in higher drug bioavailability [59].

### 3.5.2. In Vivo Evaluation of Anticancer Potential

The anticancer efficacy of different NS formulations was assessed on female mice bearing Ehrlich ascites tumors, a well-established model of spontaneous murine mammary adenocarcinoma [60]. Efficacy assessment started when tumor size reached ~100 mm3. Treatment was administered by IP injection over 14 days.

### Tumor Growth Inhibition

Figure 9A(a) demonstrates the percentage increase in tumor size for different study groups during the study period. Representative photographs of excised tumors following sacrifice are shown in Figure 9A(b). The positive control mice exhibited a significant increase in tumor size throughout the study period, with ~600% after 16 days (*p* ≤ 0.01). Tumor growth inhibitory effect was achieved following treatment with FS suspension, FS-NS, and LF-FS-NS, with a percentage increase in tumor size of 209.4, 128.9, and 116.7 %, respectively, after 16 days, which is consistent with the excised tumor images (Figure 9A(b)).

**Figure 9.** (**A**) Antitumor effect of FS and NS formulations in comparison with untreated control 16 days post-IP treatment of Ehrlich ascites mammary tumor in mice showing: (**a**) percentage change in tumor size relative to pretreatment volume and (**b**) digital images of excised tumors. (**B**) Tumor biomarker levels following 16-day treatment with FS and FS-NS formulations compared to untreated control: (**a**) cyclin D1, (**b**) Bcl-2, (**c**) Bax, and (**d**) caspase-3. Data were expressed as means ± SD (n = 3). Data were analyzed using one-way ANOVA followed by post-hoc Tukey's test for group comparisons. Means of similar symbols were statistically insignificant: a>b>c>d>e(*p* ≤ 0.05).

### Assessment of Tumor Biomarkers

Cyclins are a family of cell proteins controlling cell cycle progression and promoting tumor proliferation with cyclin D1 (CD1) being a cell-cycle regulator essential for G1 phase progression. It is overexpressed in more than 50% of breast tumors [61]. In the current study, the positive control group demonstrated a significant overexpression in CD1 level by 2.4-fold compared to the negative control group (*p* ≤ 0.001) (Figure 9B(a)). Conversely, all treated groups showed a significant reduction in CD1 level compared to the positive control (*p* ≤ 0.001) in the following order: FS suspension < FS-NS < LF-FS-NS. The FS effect on lowering CD1 transcription in breast cancer is consistent with previous reports [26]. This effect was significantly augmented for FS-NS, which could be explained by the FS bioavailability enhancing effect of NS and the improved cell penetration via the EPR effect [62,63]. The LF-FS-NS formulation achieved the lowest expression of CD1, confirming its superior cytotoxic potential.

Intrinsic apoptosis, or programmed cell death, includes initial mitochondrial perturbation arising from cytotoxicity and is mainly regulated by the Bcl-2 protein family [64]. The latter is subdivided into pro-apoptotic proteins, such as Bax, and anti-apoptotic ones, such as Bcl-2. The affinities and relative abundance of different Bcl-2 proteins control whether anti-apoptotic or pro-apoptotic reactions predominate [64]. Overexpression of Bcl-2, present mainly on the outer membrane of the mitochondria, protects against apoptosis induced by many cytotoxic agents [65]. On the contrary, the increased expression of the pro-apoptotic proteins, such as Bax, will stimulate the release of mitochondrial cytochrome C into the cytoplasm and, subsequently, the activation of caspase-3 [65]. Caspase-3, a key mediator of apoptosis, is a frequently activated death protease that catalyzes the specific cleavage of several vital cellular proteins [66]. In this context, the levels of Bcl-2, Bax, and caspase-3 biomarkers were quantitively determined in tumor tissues to investigate the possible molecular pathway for the anticancer activity of FS. As shown in Figure 9B(b,c), the positive control group showed a significant up-regulation in the levels of the anti-apoptotic

protein Bcl-2 by ~3-fold compared to the negative control group (*p* ≤ 0.001). Contrarily, FS suspension significantly decreased the levels of Bcl-2 by 13.5% and increased the expression of the Bax gene by ~2.5-fold compared to the positive control group (*p* ≤ 0.05), which is in agreement with previously reported data [22,49]. Furthermore, FS loading into NS resulted in a significant down-regulation in Bcl-2 levels by 17.6% with an up-regulation in Bax gene expression by 1.7-fold compared to FS suspension (*p* ≤ 0.05), elucidating the role of supramolecular complex formation between FS and NS in enhancing FS anticancer activity. Interestingly, LF-FS-NS significantly achieved a higher reduction in Bcl-2 levels by 26.2% and an increase in Bax gene expression (1.9-fold) vs. uncoated FS-NS (*p* ≤ 0.05).

To further confirm the apoptotic capability of different treatment groups, analysis of the caspase-3 gene was carried out. As previously reported, FS-induced apoptosis acts through mitochondrial- and caspase-3-dependent pathways [67]. As shown in Figure 9B(d), all treatments significantly increased the expression of the caspase-3 gene with variable degrees vs. the positive control group (*p* ≤ 0.05). This could be due to the proven ability of FS to up-regulate Bax gene expression and, subsequently, the induction of apoptosis via caspase-3 activation [67]. Again, the LF-coated formulation exhibited the highest elevation in caspase-3 expression, reflecting superior efficacy. This could be explained by its ability to actively target LF receptors overexpressed on breast cancer cells [28], thus, allowing for efficient FS cellular uptake and internalization, as demonstrated by the cellular uptake study (Section 3.4.4). Moreover, on the subcellular level, LF can increase drug nuclear localization, hence, achieving optimum efficacy, as the nucleus is the main site of action for most anticancer drugs [53]. This highlights the potential of NS surface modification with LF to improve the anticancer activity of FS via an active targeting mechanism. Indeed, LF-targeted formulations have previously shown higher anticancer activity compared to both free drug and untargeted nanotherapy [33,56].

### Histopathological Evaluation

Findings of the tumor growth inhibition study and tumor biomarkers were additionally verified via histopathological examination of excised tumor tissue (Figure 10). Examined sections from normal control groups (Figure 10a,b) showed normal breast tissue architecture. Glands were organized into lobules of complex branching alveolar glands with extensive interlobular connective tissue and fat between them. The stromal compartment predominated and is packed with adipocytes, which offered insulation and aided in the protection of the fragile mammary gland tissue. The ductal lobular system's cellular lining was bilayered and composed of inner (luminal) epithelial cells, which were cuboidal to columnar in shape and had a pale eosinophilic cytoplasm. Outer (basal) myoepithelium cells were variable in form, ranging from flattened cells with compressed nuclei to prominent epithelioid cells with a copious transparent cytoplasm, and could occasionally have a myoid appearance.

Conversely, the typical lobular and ductal architecture was lost in the positive control group (Figure 10c,d), which revealed the highest intraductal proliferation in the gland, as evidenced by the formation of irregular dark proliferation sites with aberrant nuclei and a mild appearance of lymphoid tissue. Moreover, there was little evidence of fibrous interductal stroma, indicating medullary cancer. Furthermore, myoepithelium loss was seen as an indication of invasion.

Treatment with FS suspension influenced the histopathological characteristics by the formation of some intralobular connective tissue but still condensed in a non-fibrous aspect with obvious proliferating ductal glands showing lumen degradation, reflecting minor efficacy (Figure 10e,f). On the other hand, treatment with different FS-loaded NS formulations presented variable effectiveness. In this respect, FS-NS accomplished lower efficacy, as indicated by the decrease in the number of proliferative cells, but aberrant stroma and many lymphocytes were still seen (Figure 10g,h). Interestingly, LF-FS-NS demonstrated the greatest anticancer potential, as indicated by the absence of any proliferative cells, but still showed disorganized cells (Figure 10i,j) owing to the short treatment time. Some

adipose tissue started to be retrieved. Nearly normal breast tissue architecture was seen. Additionally, the interlobular stroma appeared normal with many lymphocytes.

**Figure 10.** Photomicrographs illustrating H&E-stained breast tissue specimens of (**a**,**b**) normal breast and (**c**,**d**) positive control group and groups treated with (**e**,**f**) FS suspension, (**g**,**h**) FS-NS, and (**i**,**j**) LF-FS-NS. Yellow arrow: ductal system. Red arrow: adipose connective tissue. Black arrow: connective tissue stroma. Black star: degeneration. Red star: lymphocytes. The right panel represents a higher magnification of the area selected in the left panel. Magnification ×100, scale bar 200 μm (low)- ×200, scale bar 100 μm (high).

To sum up, combined results of biochemical and histopathological evaluation verified the superiority of LF-FS-NS in the treatment of breast cancer compared to both FS

suspension and uncoated formulation, suggesting successful tumor targeting and drug accumulation in cancer cells.

### 3.5.3. In Vivo Toxicity

All mice treated with FS test formulations survived and appeared healthy throughout the study. An insignificant change in body weight (*p* ≥ 0.05) was observed. Evaluation of liver and kidney functions was performed, and the results are described in Table 5. Liver enzymes, namely ALT and AST, were assessed as indicators for proper liver performance, while serum levels of both urea and creatinine were measured as indicators of renal function. Results demonstrated insignificant changes in the measured parameters for all tested groups compared to normal healthy control mice (*p* ≥ 0.05), suggesting the safety of the developed formulations. Our results were in accordance with many reported articles describing the safety and biocompatibility of ß-CD-NS [68,69].

**Table 5.** Effect of free FS suspension and NS formulations on liver and kidney function tests.


The study was conducted on female albino mice with six animals in each group (n = 6). Values were expressed as mean ± SD. Data were analyzed using one-way ANOVA followed by a post-hoc test (Tukey's) for group comparisons.

To further assess the in vivo toxicity, histopathological examination of the most affected organs (liver, kidney, and spleen) was performed at the end of treatment (Figure 11A–C). The spleen sections (Figure 11A) isolated from different study groups showed almost normal architecture, with a well-delineated white and red pulp with continuous trabecular throughout the tissues encountered by a capsule. The white pulp included lymphoid follicles as well. Nonetheless, the FS suspension group showed some dilatation and the development of bleeding (Figure 11A(c)).

Liver sections for FS-loaded NS groups demonstrated typical hepatocyte appearance with normal cells in the center having polyhedral shape, vacuolated acidophilic cytoplasm, and rounded vesicular nuclei (Figure 11B(d,e)). However, examined specimens from the FS suspension group exhibited mild inflammation near the central vein (Figure 11B(c)).

Despite the extensive reports on the nephroprotective role of FS [70], the renal tissue was the most affected by FS suspension (Figure 11C(c)). Since this was only obvious based on histopathological but not biochemical analysis, the slight changes observed could be attributed to different factors, including mice physiological state and other effects on nutrients uptake, among others [68]. Interestingly, renal tissues showed no symptoms of toxicity for FS-NS formulations (Figure 11C(d,e)). Normal appearance of (Malpighian) corpuscles was evident, which were comprised of glomerular capillaries and Bowman's capsules with subcapsular space. Many proximal convoluted tubules were lined with simple truncated cubical (pyramidal) cells with basal spherical nuclei and had narrow lumina. The lumina in distal convoluted tubules were large.

**Figure 11.** Photomicrographs illustrating H&E-stained (**A**) spleen tissue specimens of (**a**) normal group, (**b**) positive control group, (**c**) FS suspension-treated group, (**d**) FS-NS-treated group, and (**e**) LF-FS-NS-treated group. Black arrow: hemorrhage. W: white pulp. R: red Pulp. (**B**) Liver tissue specimens of (**a**) normal group, (**b**) positive control group, (**c**) FS suspension-treated group, (**d**) FS-NS-treated group, and (**e**) LF-FS-NS-treated group. Black arrow: inflammation. (**C**) Kidney tissue specimens of (**a**) normal group, (**b**) positive control group, (**c**) FS suspension-treated group, (**d**) FS-NS-treated group, and (**e**) LF-FS-NS-treated group. Magnification ×100 spleen-×200 liver and kidney.
