**1. Introduction**

Nanosponges (NS) are promising polymeric colloidal systems. As the name implies, NS are nano-sized porous structures offering ideal properties for drug delivery. NS can be synthesized from various polymers and copolymers, such as hyper cross-linked polystyrenes [1], ethyl cellulose [2], or cyclodextrins [3]. Cyclodextrins (CDs) are generally recognized as safe (GRAS), as listed by the Food and Drug Administration (FDA) [4]. Including β-CD as the most studied and most frequently used, CDs are cyclic oligosaccharides with a cage-like structure and a distinct cone-shaped lipophilic cavity [5]. This unique structure contributes to their ability to form inclusion complexes with various molecules, resulting in enhanced aqueous solubility and protection against degradation [6]. However, the ease of dissociation of the formed complexes upon dilution, in addition to the inability to complex certain molecules, are considered major drawbacks [6]. To overcome these limitations, structural modifications of CDs have been suggested to increase the inclusion capacity and allow for a wider scope of biological applications [7]. Of these, cyclodextrin-based nanosponges (CDNS) have recently attracted attention and been synthesized by polymer cross-linking to form a highly porous branched matrix [3]. The obtained amphiphilic spongy structure confers the ability to accommodate hydrophobic

**Citation:** Aboushanab, A.R.; El-Moslemany, R.M.; El-Kamel, A.H.; Mehanna, R.A.; Bakr, B.A.; Ashour, A.A. Targeted Fisetin-Encapsulated β-Cyclodextrin Nanosponges for Breast Cancer. *Pharmaceutics* **2023**, *15*, 1480. https://doi.org/10.3390/ pharmaceutics15051480

Academic Editors: Franco Dosio and Ana Isabel Fernandes

Received: 30 March 2023 Revised: 25 April 2023 Accepted: 10 May 2023 Published: 12 May 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

molecules in the CD cavities and fewer lipophilic molecules in the more hydrophilic outer polymeric network with high loading capacity and controlled drug release [8]. Furthermore, CDNS offer other advantages, such as being highly biocompatible, biodegradable, and of low cytotoxicity [9]. In addition, this nanosystem was shown to improve permeation across biological barriers and enhance the bioavailability of active substances [6]. As a controlled drug delivery system, CDNS has been explored for oral, topical, and parenteral drug delivery for numerous applications, such as anticancer, antiviral, antihypertensive and antiplatelet therapy [5,10]. One of the most relevant fields is drug delivery for cancer therapy [9,11], including breast cancer. In this aspect, CDNS showed improved anticancer drug effects both in vitro [12,13] and in vivo [14].

The breast cancer (BC) burden has been rising sharply over the past decades. Having replaced lung cancer, it is now the most diagnosed cancer worldwide, representing a quarter of all cancer cases in females. Despite the significant advances in BC treatment, a continuous rapid increase in the number of new cases and deaths is recorded, with a 40% projection expected especially in low human developing index countries by 2040 compared to 2020 [15]. This could be imputed to the limitations of chemotherapy, including non-selectivity to cancer cells, multidrug resistance (MDR), and ineffective inhibition of tumor growth, metastasis, and recurrence. Thus, recent strategies for the prevention and treatment of BC focused on the use of herbal medicine as a safe, effective, and low-cost alternative to cytotoxic drugs. Considerable anticancer activity in natural phytochemicals has been confirmed against different cancers via numerous in vitro and in vivo studies [16]. Flavonoids are plant polyphenols exhibiting various beneficial properties for breast cancer therapy [17]. They were shown to affect growth and proliferation via cell cycle arrest, necrosis, and apoptosis. Furthermore, they exhibit antioxidant, anti-inflammatory, and anti-mutagenic properties [18]. A potent flavonoid found in various fruits and vegetables and exhibiting cytotoxic activity is fisetin (3,3 ,4 ,7-tetrahydroxy flavone, FS) [19]. The anticancer effect of FS on different breast cancer cell lines including triple-negative breast cancer (TNBC) cells was manifested [20,21]. Among several mechanistic studies on different breast cancer cell lines, FS was shown to inhibit cell proliferation and metastasis, prevent cell cycle progression, induce apoptosis, cause cleavage of poly ADP ribose polymerase (PARP), and modulate Bcl-2 family protein expression [22]. Moreover, FS was capable of suppressing PKCα/ROS/ERK1/2 and p38 MAPK signaling pathway activation, reducing NF-κB activation, and lowering TET1 expression in a concentration- and time-dependent manner [22,23]. It also reverses the epithelial to mesenchymal transition (EMT) process mediated by the PTEN/Akt/GSK-3β signaling pathway [20].

Despite its bioactive potential in the prevention and treatment of different cancer conditions, the clinical applications of FS have been impeded by its highly lipophilic nature, and, hence, limited aqueous solubility [24]. Furthermore, it is rapidly metabolized, enzymatically degraded, and liable to p-glycoprotein efflux following oral administration, which leads to a short half-life and poor bioavailability [24,25]. These curtailments interfere with FS bioaccessibility, necessitating the development of novel oral drug delivery approaches, such as loading into nanosystems [26,27]. Furthermore, the nanoparticles' surface could be modified with active ligands, such as folic acid, hyaluronic acid, chondroitin sulfate, and lactoferrin, for selective and efficient accumulation at tumor sites via targeting specific receptors overexpressed on BC cells [26]. Lactoferrin (LF) is an iron-binding glycoprotein possessing a strong affinity to transferrin receptors overexpressed on breast cancer cells [28]. It is capable of either promoting or inhibiting cell proliferation and migration depending on whether the cell it acts upon is normal or cancerous, respectively [29]. Moreover, being a part of the innate immune system, LF boosts adaptive immune response, thus, preventing or inhibiting cancer development [29].

In this regard, the current study aimed to develop FS-loaded cyclodextrin nanosponges (FS-NS) coated with the bioactive protein LF to enhance FS bioavailability and anticancer activity. This novel nanosystem would provide multiple advantages, including high drug loading and entrapment efficiency with sustainment of release and an improvement in the

anticancer activity of FS via both passive targeting by enhanced permeability and retention (EPR) effect and active targeting by the LF coating. Following formulation optimization and in vitro characterization, the change in FS pharmacokinetic parameters upon loading into proposed LF-FS-NS relative to FS suspension and uncoated FS-NS was assessed. Moreover, the cytotoxicity and cellular uptake of the test formulations were assessed on MDA-MB-231, a human TNBC cell line. A murine Ehrlich ascites breast cancer mouse model was used to assess in vivo anticancer efficacy.

### **2. Materials and Methods**

### *2.1. Materials*

β-cyclodextrin, diphenyl carbonate (DPC), coumarin 6 (C6), and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lactoferrin (LF) was obtained from Lactoferrin.co (Frankfurt, Germany). Fisetin (FS) was from Arctom Scientific (Agoura Hills, CA, USA). HPLC-grade acetonitrile, methanol, dimethylformamide (DMF), and formic acid were purchased from Fischer Scientific (Loughborough, UK). Hoechst 33342 stain, an annexin V FITC/propidium iodide (PI) kit, fetal bovine serum (FBS), and Dulbecco's modified Eagle's medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin and streptomycin solution (100 U/mL each) were from BioWhittaker® (Lonza, Belgium). 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was purchased from Serva (Heidelberg, Germany). PCR primers were obtained from Eurofins Scientific (Luxembourg). A one-step RT qPCR kit (SYBR Green with low ROX) was from Enzynomics Co. Ltd., Yuseonggu, Daejeon, Korea. All the other reagents were of analytical grade and were used without further purification.

### *2.2. Preparation of Blank Nanosponges*

NS formulations were prepared using diphenyl carbonate (DPC) cross-linker as previously described, with some modifications [30]. In brief, β-cyclodextrin (CD) was dissolved in N, N-dimethylformamide (DMF), and DPC was added in a molar ratio of 1:6. The reaction mixture was mixed under magnetic stirring at 450 rpm for 20 min in a water bath at 80 ◦C. For optimization, the effects of heating the reaction mixture at 90, 120, or 150 ◦C for different time intervals (2 or 5 h) while using different volumes of DMF (3 or 6 mL) were investigated. The product was subjected to 5 cycles of washing with deionized water and acetone and then left to dry in a desiccator for 48 h. The purified powdered formulation was ground in a mortar and accurately weighed to calculate the percentage yield using the following equation:

$$\% \text{ Yield} = \frac{\text{Weight of the nanosecond}}{\text{Weight of } \beta - \text{CD} + \text{Weight of DPC}} \times 100\tag{1}$$

The obtained powder (14 mg) was dispersed in 2 mL deionized water and sonicated using a probe sonicator (Bandelin Sonoplus, Germany) at 60% amplitude for 10 min. Homogenization at 10,000 rpm for 10 min was subsequently carried out to obtain a nanodispersion (NS).

### *2.3. Ferric Chloride Test*

To qualitatively verify NS formation, the FeCl3 test was performed to detect the presence of phenols formed as a by-product during the cross-linking esterification reaction between β-CD and DPC. Unwashed NS (10 mg) were dispersed in 3 mL of deionized water followed by the addition of 1 mL FeCl3 solution. The appearance of a deep violet color indicated the presence of phenol, proving NS formation [30].

### *2.4. Preparation of Fisetin-Loaded Nanosponges*

For the preparation of fisetin-loaded nanosponges (FS-NS), the drug was dissolved in ethanol and added to the NS dispersion in a ratio of 1:4 to obtain a final drug concentration of 1.75 mg/mL. For complete drug loading, the mixture was subjected to 5 min sonication in a bath sonicator followed by overnight stirring [10].

### *2.5. Preparation of Lactoferrin (LF)-Coated FS-NS*

FS-NS formulation dispersion (2 mL) was dropped into 100 μL of LF solution in PBS pH 6.5 and stirred at 500 rpm for 30 min. Different LF concentrations (25–100 mg/mL) were tested. Efficient LF coating was verified by size and ζ-potential measurements.

### *2.6. Physicochemical Characterization*

### 2.6.1. Surface Area and Porosity Analysis

Specific surface area and porosity of the prepared NS were measured using a nitrogen absorption–desorption isotherm (Belsorp-Mini II analyzer, Japan). The sample (0.2 g) was degassed for 3 h before analysis. The surface area and porosity were determined using Brunauer–Emmett–Teller (BET) and Barrett–Joiner–Halenda analyses [31].

### 2.6.2. Microscopical Examination

### Scanning Electron Microscopy (SEM)

The surface morphology and porosity of blank NS were evaluated using a scanning electron microscope (SM-IT200; JEOL, Tokyo, Japan). The NS dispersion was mounted on a metal stub, air-dried, and sputter-coated with gold before the examination.

### Transmission Electron Microscopy (TEM)

Transmission electron microscopy (TEM) (model JEM-100CX, JEOL, Japan) was used to further investigate the morphology as well as to determine the average size of NS, FS-NS, and LF-FS-NS [32]. Dispersions were dropped on carbon-coated copper grids, stained with uranyl acetate (1% *w*/*v*), and air-dried before the examination. For the determination of particle size (PS), 50 measurements from different fields were carried out using imageanalysis software [31] (Fiji 1.52p; National Institutes of Health, Bethesda, MD, USA) and the polydispersity index (PDI), calculated by the following equation:

$$\text{PDI} = \left(\frac{SD}{d}\right)^2\tag{2}$$

where *SD* is the standard deviation and *d* is the average diameter.

### 2.6.3. ξ-Potential Measurement

ζ-potential of different formulations (NS, FS-NS, LF-FS-NS) was determined using a Malvern Zetasizer (Nano-ZS Series DTS 1060, Malvern Instruments, UK). Measurements were performed at a fixed angle of 173◦ at 25 ◦C. Formulations were adequately diluted with deionized distilled water in the ratio 1:100 and measured in triplicate.

### 2.6.4. Entrapment Efficiency and Drug Loading Determination

Fisetin entrapment efficiency% (EE%) was carried out using the dialysis technique [33]. FS-NS and LF-FS-NS dispersions (0.5 mL) were placed in a dialysis bag (Visking 36/32, 28 mm, MWCO 12–14 KDa; Serva, Heidelberg, Germany) and then immersed in 26 mL PBS (pH 7.4) containing 0.1% Tween® 80 to maintain sink conditions, before being centrifuged at 25 ◦C and 500 rpm for 30 min using a high-speed cooling centrifuge (Model 3K-30; Sigma Laborzentrifugen GmbH, Osterode, Germany). The free unentrapped drug in the eluent was quantified spectrophotometrically at 360 nm using a UV–visible spectrophotometer (Cary 60 UV–visible spectrophotometer, Agilent, Santa Clara, CA, USA). Linearity was checked in the range 5–15 μg/mL with a coefficient of determination R<sup>2</sup> = 0.99. For % drug loading (DL%) determination, NS was redispersed in ethanol following centrifugation. The

amount of FS analyzed denoted the weight of FS in NS formulations [10]. EE% and DL% were calculated using the following equations:

$$\text{EE }\% = \frac{\text{Total drug amount} - \text{ unentraped drug amount}}{\text{Total drug amount}} \times 100\tag{3}$$

$$\text{DL }\% = \frac{\text{Drug amount in NS}}{\text{Total weight of NS}} \times 100\tag{4}$$

### 2.6.5. In Vitro Drug Release

In vitro release of FS from both FS-NS and LF-FS-NS in comparison to the drug solution was carried out using the dialysis bag method. The drug solution was prepared in an aqueous PEG 400 solution (50% *v*/*v*). NS dispersions (0.5 mL equivalent to 0.9 mg FS) were added into dialysis bags and then placed into 30 mL PBS (pH 7.4) with 0.1% *w*/*v* Tween® 80, maintaining sink conditions. The experiment was carried out in a thermostatically controlled shaking water bath at 37 ◦C and 100 rpm. At different time intervals (1–24 h), samples (1 mL) were withdrawn and replaced with fresh medium. The drug concentration was determined spectrophotometrically at 360 nm. Then, the percentage of cumulative FS released was calculated in triplicate.

Drug release kinetics from NS dispersions were assessed using model-dependent methods [34] calculated by the Excel add-in DDsolver [35].

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

Prior to analysis, FS-NS dispersion was dried using Aerosil® 200, as previously described [36]. Briefly, FS-NS aqueous dispersion was mixed with Aerosil® 200 in a ratio of 4:1, and then the mixture was allowed to dry overnight in a desiccator. The FTIR spectra of Aerosil® 200, β-CD, DPC, NS, FS, and FS-NS were obtained using an FTIR spectrometer (PerkinElmer Inc., Waltham, MA, USA). Tested samples were mixed with KBr (1:100 *w*/*w*) and compressed to form a disc that was scanned in the range 4000–500 cm<sup>−</sup>1.

### 2.6.7. X-ray Powder Diffractometry (XRD)

The crystallinity of Aerosil® 200, NS, FS, and FS-NS was assessed using XRD (XRD-7000 X-ray diffractometer, Bruker D2-Phaser; Madison, WI, USA). The diffraction pattern was performed in a step scan model of 30 kV and 30 mA with a scanning region for the diffraction angle, 2θ, from 0 to 100◦, with step size 0.02◦.
