Preparation and Characterization of MUC-30-Loaded Polymeric Micelles against MCF-7 Cell Lines Using Molecular Docking Methods and In Vitro Study

MUC-30 is a hydrophobic compound which is active against the MCF-7 cancer cell line. In this study, MUC-30 was loaded in polymeric micelles to improve the water solubility and release rate. For prolonged MUC-30 release, MUC-30 was encapsulated in polymeric micelles using PEG-b-PLA and PEG-b-PCL as materials. Micelles prepared with 1 : 9 w per w ratios by film hydration achieved the highest entrapment efficiency (EE%). The EE% of MUC-30-loaded PEG-b-PCL micelles was approximately 30% greater than that of PEG-b-PLA micelles, due to the different H-bond formations between MUC-30 and the polymer membrane (PCL, 4; PLA, 3). The cytotoxic activity of MUC-30 against EGFR theoretically presented 399.31 nM (IC50 = 282.26 ng/mL) by molecular docking. In vitro cytotoxic activity of MUC-30 was confirmed by MTT assay. MUC-30 (IC50 = 11 ± 0.39 ng/mL) showed three-fold higher activity over MUC-30-loaded PEG-b-PLA micelles (IC50 = 37 ± 1.18 ng/mL) and two-fold higher over PEG-b-PCL micelles (IC50 = 75 ± 3.97 ng/mL). This was due to the higher release rate of MUC-30 from PEG-b-PLA micelles compared to PEG-b-PCL micelles. Therefore, MUC-30-loaded PEG-b-PLA micelles could be a promising candidate for breast cancer chemotherapy.


Introduction
Worldwide, in 2018, the most common cancer in women was breast cancer, with approximately 2.1 million cases [1]. Most breast cancer deaths are due to migration of the tumor to other parts of the body and the complexity of molecular mechanism. e effectiveness of MUC-30 lies in its ability to bind with targeted proteins to overcome these limitations involving in the development and growth of breast cancer caused from drug-resistant mechanisms [2]. Breast cancer usually presents the following proteins: estrogen receptor (ER) [3], progesterone receptor (PR) [4], epidermal growth factor receptor (EGFR) [5], and human epidermal growth factor receptor 2 (HER2) [6]. In addition, proteins that are related to drug resistance are P-glycoprotein (Pgp) [7] and NF-κB activation [8]. erefore, diminishing the expression of ER, PR, EGFR, HER2, Pgp, and NF-κB should be an important strategy to inhibit the growth and drug resistance of breast cancer cells. To determine protein inhibition, binding affinity of this compound to the mentioned proteins will be evaluated and compared to standard treatment such as tamoxifen [9]. MUC-30 ( Figure 1), a semisynthetic analog of cleistanthin A from Phyllanthus taxodiifolius Beille, can be utilized to inhibit breast cancer [10]. Nevertheless, the use of MUC-30 has limitations, i.e., poor water solubility and multidrug resistance (MDR) caused to induce expression of P-glycoprotein (Pgp) and NF-κB activation. To overcome these obstacles, a polymeric micelle from block copolymers was employed to encapsulate MUC-30 within the core. Encapsulation of drugs in these block copolymeric micelles including poly(ethylene glycol)-b-poly (D, L-lactide) (PEG-b-PLA) and poly(ethylene glycol)-b-poly (ε-caprolactone) (PEG-b-PCL) [11] has been proved to increase water solubility of drugs [12] and prevent the development of drug resistance inhibiting ABC-transporter-mediated drug efflux [13][14][15]. ese micelles were proved to be safe in animals [16]. erefore, in this work, we evaluate MUC-30-loaded polymeric micelles' properties associated with water solubility, drug entrapment, drug release, and the ability of MUC-30 to inhibit MCF-7. Moreover, targeted proteins relating to breast cancer such as ERα, PR, EGFR, HER2, Pgp, and NF-κB were analyzed for impact after being treated with MUC-30 by the estimation of IC 50 values calculated by AutoDock [17]. Results were compared to IC 50 obtained by MTT assay [18][19][20].

Cell Line.
A human breast adenocarcinoma cell line (MCF-7) was purchased from the American Type Culture Collection to be used in the cytotoxicity test. It was cultured by the DMEM (Dulbecco's Modified Eagle's medium), which was obtained from Gibco (Grand Island, New York). Most cancer cell lines with the DMEM were able to obtain better growth than the Minimum Essential Medium (MEM) due to the DMEM having four times the number of vitamins and amino acids and 2-fold of glucose. e supplemental agents 10% fetal bovine and 1% penicillin/streptomycin (pen/strep) were purchased from JR Scientific Inc. (Woodland, California) and added to the DMEM. e MCF-7 cell line was cultured in an incubator with a human-like environment at 5% CO 2 in humidified atmosphere at 37°C.

Water Solubility.
e solubility of MUC-30 can also be predicted computationally using the mathematical software COSMOquick. e COSMOquick approach uses a QSPR technique [21] to estimate solubility. In this study, ΔG fus has been calculated according to the following equation: where ΔH fus is the enthalpy of fusion, T is set at room temperature, and T m is the melting temperature for MUC-30. ese values were estimated efficiently using COSMOquick.

Molecular Modeling.
e targeted protein structure of ERα (PDB code: 3ERT), PR (PDB code: 4OAR), EGFR (PDB code: 2J6M), HER2 (PDB code: 3WSQ), Pgp (PDB code: 6QEX), and NF-κB (PDB code: 1SVC) was collected from the Protein Data Bank. e structure of the MUC-30 ligand is as given in Figure 1 which was drawn in ChemSketch 3.5; then, MUC-30 was submitted to the energy minimization tool using Arguslab software [22]. e geometry of MUC-30 was optimized using the semiempirical (PM3) Hamiltonian with Restricted Hartree-Fock (RHF). Both the ligand and targeted proteins were prepared in a PDB format prior to docking using Avogadro software [23].

Building Polymer Surface.
Monte-Carlo and molecular dynamics methods were utilized for constructing polymers with surfaces. e polymer structure was optimized using energy constraints. e polymer surface [24] was prepared following a confined surface of PLA at a density of 1.27 g·cm − 3 and PCL at a density of 1.15 g·cm − 3 . PLA and PCL with twenty-five repeating units were reconstructed in an orthorhombic cell of dimension 36Å × 36Å × 18.8Å and 36Å × 36Å × 18.2Å, respectively.

Binding Site Analysis.
After docking, the docked complexes were visualized in the Discovery Studio to investigate MUC-30 interactions with targeted proteins and the polymer surface [25,26]. e results were presented in two types: (1) the binding site of the targeted protein for docking is specified. (2) e MUC-30 ligand was docked to the specified prepared polymer surface which is the PLA and PCL surface.

Preparation of MUC-30-Encapsulated Polymeric
Micelles. MUC-30-loaded polymeric micelles were fabricated by the film sonication method [12,27]. MUC-30 and the polymer were dissolved in tetrahydrofuran (THF). To obtain the film, the solvent dissolving mixture was evaporated by using a rotary vacuum evaporator (IK, RV10). After that, distilled water was added to film and subsequently sonicated for 1 min by using Sonic-VibraCell ™ (model CV.18, 130 W, 20 kHz).

Particle Size Determination.
MUC-30-loaded polymeric micelles were prepared through film sonication; then, the size and size distribution of total MUC-30 entrapped in the polymeric micelles at the concentration of 2 mg·mL − 1 were determined by laser light scattering (Zetasizer Nano ZS, Malvern).

Water Solubility of MUC-30.
UV-Vis spectroscopy was utilized to determine the solubility of the compounds. Firstly, 2.5 mL of THF was used to dissolve 1 mg of MUC-30. e dissolved solution was dropped into 100 mL of water and stirred for 72 h at room temperature to allow THF evaporated [27]. e insoluble drug was removed by refrigerated centrifugation (4°C) for 10 min at 3000 rpm and 0.45 μm syringe filtration. e solution was then lyophilized. e water solubility of MUC-30 was calculated from the total volume of water added after being dissolved in 5 mL of DMSO.

Drug Loading Study.
e amount of MUC-30 encapsulated in polymeric micelles was determined by UV-Vis spectroscopy. e freshly prepared micelle solution (10 mL) was purified by refrigerated centrifugation for 10 minutes at 3000 rpm and filtration through a 0.45 μm syringe filter to remove polymer aggregation. After purifying, there are still some unencapsulated drugs. erefore, a centrifugal filter with a 50 kDa molecular weight cutoff (Millipore, USA), which could separate the drug-loaded micelles located above while unencapsulated drugs fall, was employed to remove unencapsulated MUC-30. e unencapsulated MUC-30 was collected and lyophilized. Lyophilized particles were subsequently measured for the number of MUC-30 by dissolving in chloroform. e lyophilized micelles were also measured by dissolving in chloroform. e absorbance of MUC-30 was recorded at 263 nm. e drug properties such as drug loading density, drug loading efficiency, and yield were calculated using the following equations: % drug loading density � the amount of drug in micelles the amount of micelles − free drug × 100, % drug loading efficiency � the amount of drug in micelles the initial amount of drug in system × 100, % yield � total micelle amount of drug − free drug theoretical total amount of micelle × 100.  e release studies were performed at 37°C. At a predetermined time, PBS was taken to measure the amount of MUC-30 at selected time intervals and 20 mL of fresh PBS was replaced [28]. e amount of MUC-30 in a cosolvent of PBS and ethanol was detected using a microplate reader at the excitation wavelength at 260 nm and the emission wavelength at 418 nm. To explain the drug dissolution process, the drug release data were computed using DDsolver [29].

In Vitro Cytotoxicity Test.
e in vitro cytotoxicity test was carried out in the Laboratory for Biocompatibility Testing of Medical Devices, Mahidol University. MCF-7 was evaluated by MTT assay. A density of MCF-7 at 5 × 10 3 cells per 100 μL medium was seeded into 96-well plates. After 1-day incubation, cells were washed once with the medium and the various MUC-30 concentrations prepared in the medium were added. e cell viability was evaluated 72 h after the treatment by MTT assay. Cell survival was defined by a change in the cell color to bluepurple and was conducted by dissolving formazan in dimethyl sulfoxide (DMSO). e color intensity was recorded at 570 nm using a microplate reader (TECAN).

Statistical Analysis.
All experiments were carried out in triplicate and are presented as the mean ± standard deviation (SD). An analysis of the data (ANOVA) was conducted using SPSS Statistics 17.0. A p value which was less than 0.05 was considered to be statistically significant.
is is due to aggregation of MUC-30-loaded PEG-b-PLA at a ratio of 3 : 7.

Size and Drug Loading Content of MUC-30 in Micelles.
All ratios of MUC-30-loaded polymeric micelles provide a proper size of micelles (10-200 nm). Data show that the amount of MUC-30 loaded did not affect the size, as shown in Table 1. e large amount of MUC-30 loaded in PEG-b-PCL micelles provides smaller particle size than that of MUC-30-loaded PEG-b-PLA micelles. is was due to poor water-soluble property of the structure, leading to greater swelling of MUC-30-loaded PEG-b-PLA particles. erefore, particle size was mainly affected by the type of copolymer. e PCL structure encapsulated larger amount of MUC-30 than PLA. MUC-30 and the polymer were selected for further experiments including the release profile and cytotoxicity because this ratio provides the smallest size and high drug loading including drug loading density, encapsulation efficacy, and yield [30]. is study suggested that the ratio at 1 : 9 w per w of MUC-30 and polymer is the proper proportion to encapsulate MUC-30.

In Vitro Release Study.
e in vitro release study in PBS at pH 7.4 and 37°C shows that MUC-30 released slower as a result of the PCL structure than the PLA structure and also caused by higher hydrophobicity of PCL compared to PLA, as shown in Figure 3. is is similar to other studies that hydrophobic compounds had a slower release rate than hydrophilic compounds [27,31].
Eight release models including Baker-Lonsale, first order, Hopfenberg, Hixson-Crowell, Higuchi, Korsmeyer-Peppas, quadratic, and zero order were fitted to find the best-fitted release model and explain the mechanism of drug release. e best-fitting model was the Korsmeyer-Peppas model with R 2 > 0.99 (Tables 2 and 3), with the release of MUC-30 from micelles explained via the following equation: where Mt/Mα is the fraction of cumulative drug release at a specified time, k is the constant of drug release rate, n is employed to explain the release mechanisms such as diffusion or polymer relaxation and combination mechanisms between diffusion and erosion control.

Computational Calculation of Toxicity Using AutoDock.
In this study, binding affinities between MUC-30 and targeted proteins that are overexpressed in breast cancer were proven by docking calculations. e estimated value of binding is presented from the best binding affinity energies (kcal·mol − 1 ). MUC-30 was docked following the binding affinities with the targeted proteins EGFR, PgP, PR, NF-kB, ERα, and HER2. MUC-30 which is a semisynthetic    Table 4. Amongst the 6 targeted proteins, 2 targeted proteins that exhibited with energy values above tamoxifen binding for breast cancer receptors were EGFR and NF-kB. erefore, breast cancer can be strongly inhibited by MUC-30 due to the inhibition of EGFR and NF-kB.

Cytotoxicity Study of MUC-30 and MUC-30-Loaded Polymeric Micelles.
ese block copolymers were tested to determine the nontoxicity level with normal cells. Fibroblast cells (L929) were treated with blank polymeric micelles, PEG-b-PLA and PEG-b-PCL micelles, and compared to a normal medium. Results demonstrate that these block copolymers are biocompatible, as shown in Figures 4(a) and 4(c). e cytotoxicity of MCF-7 after treatment with MUC-30 and MUC-30-loaded polymeric micelles was measured using an MTT assay and compared to the computational data. Results show that unencapsulated MUC-30 and encapsulated MUC-30 altered MCF-7 morphology as indicated by the circular shape as shown in Figure 4 is was because of controlled release of MUC-30 from micelles.
MUC-30 has a strong binding to PCL leading to the gradual release of MUC-30 from micelles compared to PLA which was not as strong as PCL. is leads to a faster release rate of MUC-30 from PLA compared to PCL which is consistent to the number of hydrogen bondings and hydrophobic interactions between MUC-30 and the polymer surface of PCL and PLA as shown in Figure 5, causing approximately two times more toxicity.

Conclusions
However, MUC-30 was somewhat more cytotoxic than MUC-30-loaded micelles, probably because MUC-30 can be transported into the nucleus of cells by the passive diffusion mechanism, while the drug-loaded micelles have to be internalized by endocytosis, release the loaded drugs, and then, diffuse through the endocytic before entering to nucleus of cells [32]. However, the properties of the drug-loaded nanoparticles were found to be safer with normal cells (L929) than those used with free drugs [33], which results in normal cell death as well. Although the effect of drug-loaded micelles suppressing cancer cells is less than that of a free drug, it was found that the inhibitory effectiveness of MUC-30-loaded micelles was still rather close to that of MUC-30 and did not affect normal cells [33]. For achieving minimum effective dose (MEC) in vivo, it was found that the drugloaded micelles were more effective than the free drug. is is due to reduced drug clearance from the body [34] and efficiency in targeting cancer cells better through the EPR process [35]. is study has improved the MUC-30 properties and described the properties of encapsulated MUC-30 by computer simulation and in vitro experiment. Water solubility of MUC-30 was improved by encapsulation inside the micelle core and still inhibited MCF-7 growth. e release rate of MUC-30 through the micelle surface of PCL (k � 2.753; 4 HB; 7 HI) was slower than that of PLA (k � 7.236; 3 HB; 6 HI). In the cytotoxicity test, free MUC-30 (11 ng·mL − 1 ) displayed higher MCF-7-inhibiting ability than MUC-30-loaded micelles. is was probably because MUC-30 was prolonged and gradually released from polymeric micelles following the Korsmeyer-Peppas model. e higher MUC-30 loading in PEG-b-PCL micelles inversely provided smaller particle size than PEG-b-PLA micelles which was most likely due to the strong interaction between MUC-30 and PCL. Finally, we believe understanding how MUC-30 interacts with a polymer and inhibits specific proteins will help the development of the hydrophobic natural compounds.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no known conflicts of interest.