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Folate conjugated vs PEGylated phytosomalcasein nanocarriers for codelivery of fungal-and herbal-derived anticancer drugsShaymaa W El-Far1,2, Maged W Helmy1,3, Sherine N Khattab1,4, Adnan A Bekhit1,5,6,Ahmed A Hussein2 & Ahmed O Elzoghby*,1,7,8

1Cancer Nanotechnology Research Laboratory (CNRL), Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt2Department of Biotechnology, Institute of Graduate Studies & Research, Alexandria University, Alexandria 21526, Egypt3Department of Pharmacology & Toxicology, Faculty of Pharmacy, Damanhour University, Damanhour 22516, Egypt4Department of Chemistry, Faculty of Science, Alexandria University, Alexandria 21321, Egypt5Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt6Pharmacy Program, Department of Allied Health, College of Health Sciences, University of Bahrain, PO Box 32038, Manama,Kingdom of Bahrain7Department of Industrial Pharmacy, Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt8Department of Medicine, Division of Engineering in Medicine, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA02115, USA*Author for correspondence: [email protected]

Aim: Monascin and ankaflavin, the major fractions of the fungal-derived monascus yellow pigments, wereincorporated with the herbal drug, resveratrol (RSV) within the core of folate-conjugated casein micelles(FA–CAS MCs, F1) for active targeting. PEGylated RSV-phospholipid complex bilayer enveloping casein-loaded micelles (PEGPC–CAS MCs) were also developed as passive-targeted nanosystem.Results: FA– andPEGPC–CAS MCs demonstrated a proper size with monomodal distribution, sustained drug release profilesand good hemocompatibility. The coloaded MCs showed superior cytotoxicity to MCF-7 breast cancercells compared with free drugs. Both nanosystems exerted excellent in vivo antitumor efficacy in breastcancer bearing mice with PEGylated MCs showing comparable tumor regression to folate-conjugated MCs.Conclusion: Evergreen nanoplatforms coloaded with monascus yellow pigments and RSV were effectivefor breast cancer treatment.

First draft submitted: 5 January 2018; Accepted for publication: 4 April 2018; Published online:29 June 2018

Keywords: casein micelles • folate targeting • PEGylated phytosomal bilayer

Searching behind nature by the aid of nanobiotechnological approaches represented a powerful strategy in cancertreatment by improving the efficacy of antineoplastic drugs along with reducing their side effects. Throughoutthe ages, humans have depended on nature to acquire their principle requirements [1–3]. Resveratrol (RSV) is oneof the most appealing natural phytoalexin antineoplastic drugs by virtue of its apoptotic, antiangiogenesis andaromatase inhibition effect [4]. RSV inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and regulatescell differentiation, growth and proliferation. It is a potent sensitizer that affects several mechanisms involved incarcinogenesis (e.g., p53, NF-kB, PKC and cyclooxygenases [COX]) [5].

On the other avenue, microbial-derived anticancer drugs era was early began, when Fleming discovered penicillinfrom the filamentous fungus, Penicillium notatum that directed the attention to a new field in medicine focusing onantitumor antibiotics. Blenoxane R© (Nippon Kayaku Co. Ltd, Tokyo, Japan) is an example of bleomycin formulationfor treatment of squamous cell carcinoma, non-Hodgkin’s lymphoma and testicular carcinoma [6]. Monascin (MNS)and ankaflavin (ANK) are fungal secondary metabolites that possess antioxidative and antineoplastic effects. TheUS FDA has already approved Monascus red yeast extract (ANKASCIN 568-R) as a safe dietary ingredient formanaging blood sugar and improving Alzheimer’s disease symptoms [7,8]. Certainly, MNS and ANK enhancedprogrammed cell death via caspase-3 upregulation [9]. Furthermore, there is a growing evidence on the dockingeffect of MNS and ANK with aromatase enzyme receptors, which play key role in breast cancer growth [10].

Nanomedicine (Lond.) (Epub ahead of print) ISSN 1743-588910.2217/nnm-2018-0006 C© 2018 Future Medicine Ltd

Research Article El-Far, Helmy, Khattab, Bekhit, Hussein & Elzoghby

Despite their highly hydrophobic nature resulting in poor bioavailability, there were no reports about deliverysystems for MNS and ANK. Similarly, the low aqueous solubility of RSV is perceived as a major hindrance in itspotential medical application for cancer management. Polymeric micelles (MCs) hold great promise by entrappinglipophilic agents in their hydrophobic core [11–13]. Casein (CAS), the main milk protein, demonstrated amphiphilicnature being composed of both hydrophobic and hydrophilic amino acids. Thus, it is capable to self-assemble intospherical MCs [14,15].

A fundamental drawback for cancer treatment is its nonselective nature to such tumor cells. Hence, inducinguptake selectivity in tumor site by targeted delivery of the drugs acquired a significant value in cancer therapy [16].The overexpression of certain receptors on cancer cell surface (e.g., folic acid [FA] receptors) represented a keyfactor in cancer processes such as tumor invasion, metastasis, recurrence and chemoresistance. FA-conjugatedprotein–polyelectrolyte complex for targeted delivery of 5-fluorouracil against MCF-7 and MDA-MB-231 cellswas prepared by Anirudhan and Christa for targeting overexpressed folate receptors by tumor cells [17,18].

Nanophytomedicine approach could improve the bioavailability and efficacy of several herbal components.Green select phytosome, a lecithin formulation of a caffeine-free green tea catechin exerted high bioavailabilityof catechin and associated with antiproliferative effects on breast cancer tissue [19]. Moreover, PEGylation ofnanoparticles by incorporating or adsorbing of polyethylene glycol (PEG) chains on the nanoparticles’ surface actsas a stabilizing factor, contributing greatly to their antitumor efficacy. PEGylated surfaces can protect nanocarriersfrom opsonization and reticuloendothelial system clearance by repelling plasma proteins. Therefore, allowing passiveaccumulation in tumor site by enhanced permeability and retention effect (EPR) [20].

Different hybridization techniques have been utilized to formulate protein–lipid nanohybrids in order to deliverthe bioactive agents across tumor sites [21]. In our laboratory, we have developed multicompartmental PEGylatedcelecoxib (CXB)–phospholipid complex bilayer-enveloping protamine nanocapsules (NCs) to codeliver letrozole(LTZ) and CXB against breast cancer. The NCs demonstrated effective antitumor activity at the level of breastcancer cells and animal models [22,23]. According to our recent work, CAS MCs demonstrated a great potential forcombined delivery of the fungal-derived monascus yellow pigments (MYPs) and RSV in breast cancer therapy [24].Therefore, herein we investigate the influence of both passive and active targeting in enhancing their antitumorefficacy.

Therefore, in the current study, a mixture of natural antineoplastic bioactive principles (herbal RSV and fungalMYPs) was formulated in evergreen nanodelivery systems against breast cancer. First, we have developed FA-conjugated CAS MCs (FA–CAS MCs) entrapping both RSV and MYPs as actively-targeted single-compartmentnanocarriers, to enhance their uptake within breast cancer cells, via ligand-receptor-binding theory. In the secondapproach, we constructed multicompartmental nanovehicles where the hydrophobic MYPs were encapsulated intothe hydrophobic core of CAS MCs and enveloped by a bilayer of PEGylated RSV–phytosomal complex as passivelytargeted nanodelivery system. The fabricated nanovehicles were thoroughly investigated in vitro and in vivo toprove the antitumor superiority of the multiple drug nanodelivery compared with the free drug combination.

Materials & methodsMaterialsMNS and ANK were isolated from red mold rice in Microbial Biotechnology Laboratory, Institute of Grad-uate Studies and Research, Alexandria University, Alexandria, Egypt. RSV was purchased from Xi’an Nat-ural Field Bio-Technique Co. Ltd (Xian Shaanxi, China). CAS from bovine milk, FA, DMSO, N,N’-diisopropylcarbodiimide (DIC), ethyl cyanoglyoxylate-2-oxime (Oxyma), triethylamine, tertiary butyl alcohol,acetic acid 99.9% technical grade, fetal bovine serum, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bro-mide (MTT), mannitol, Triton X100, hematoxylin solution, eosin solution and Canada balsam were purchasedfrom Sigma-Aldrich (MO, USA). Fat-free soybean phospholipid with 70% phosphatidylcholine (Lipoid S75) and[(N-carbonyl-methoxypolyethyleneglycol-2000)-1,2-distearoyl-sn-glecero-3-phosphoethanolamine, sodium salt](PEG2000-DSPE) were kindly provided by Lipoid GmbH (Ludwigshafen, Germany). Tween 80 was purchasedfrom ADWIC, El-Nasr Pharmaceutical Chemicals Co. (Cairo, Egypt). Acetonitrile HPLC grade was purchasedfrom JT Baker (NJ, USA).

Preparation of FA–CASConjugation of CAS and FA was performed via carbodiimide-coupling reaction [25,26]. FA (0.088 g, 0.2 mmol)was dissolved in 10 ml DMSO. The reaction medium was rendered alkaline by addition of triethylamine (34 μl,

10.2217/nnm-2018-0006 Nanomedicine (Lond.) (Epub ahead of print) future science group

Folate conjugated vs PEGylated phytosomal casein micelles Research Article

Table 1. Composition and physicochemical characteristics of freeze-dried micellar nanosystems.Nano-formulation

CAS (mg) FA–CAS(mg)

RSV (mg) MYPs (mg) L-S75 (mg) DSPE–PEG2000(mg)

Yield (%w/w)

Particlesize (nm)

Zetapotential(mV)

PDI DL (%w/w)

EE (% w/w)

FA–CAS(blank)

– 100 – – – – 97.12 ± 1.2 124 ± 3.0 -19 ± 1.9 0.31 ± 0.02 – –

FA–CASMCs (F1)

– 100 10 10 – – 95.71 ± 2.1 137 ± 5.0 -21 ± 1.9 0.27 ± 0.02 RSV = 1.80MNS = 2.2ANK = 2.04

RSV = 86.0MNS = 86.3ANK = 85.82

PEGPC–CAS MCs(F2)

100 – 10 10 70 70 96.1 ± 3.2 272.6 ± 7.0 -36.8 ± 2.1 0.21 ± 0.03 RSV = 1.99MNS = 1.7ANK = 1.8

RSV = 99.0MNS = 76.3ANK = 78.5

ANK: Ankaflavin; CAS: Casein; DL: Drug loading; EE: Entrapment efficiency; FA: Folic acid; MC: Micelle; MNS: Monascin; MYP: Monascus yellow pigment; PC: Phospholipid complex; PDI:Polydispersity index; PEG: Polyethylene glycol; RSV: Resveratrol.

0.24 mmol), (pH 9.5). Oxyma (0.034 g, 0.24 mmol) and DIC (37.2 μl, 0.24 mmol) were then added ascoupling reagents, FA was preactivated for 5 min, followed by gradual addition of CAS (0.4 g, 0.02 mmol) tothe mixture under magnetic stirring. This interaction was left for 2 days under dark condition. The resultingFA–CAS conjugate was dialyzed by a membrane (Visking R© 36/32, 28 mm, molecular weight cut-off [MWCO]12,000–14,000, SERVA, Germany) against DMSO for 24 h to allow equilibration. The free unconjugated FA wasdetermined using a T80 UV/VIS spectrophotometer (PG Instruments Ltd, Lutterworth, UK). The absorbance(wavelength = 359 nm) of 24 h dialyzed DMSO was converted into an FA concentration by using a calibrationcurve constructed with standard FA solutions in DMSO [27]. Furthermore, this mixture was dialyzed againstincreasing gradients of DMSO:H2O and replaced every 6–8 h (100% DMSO, 90:10, 70:30, 50:50, 30:70, 10:90,100% H2O) until complete elimination of DMSO and other reaction side products. After freeze-drying, FA–CASconjugate was obtained as yellow powder.

The conjugation efficiency of FA reacted with CAS was determined by the indirect method. The unreacted freeFA found in reaction dialysate was measured by T80 UV–visible (UV–Vis) spectrophotometer (PG InstrumentsLtd) at 359 nm and calculated according to a preconstructed FA calibration curve. The UV spectra for FA, CASMCs and FA–CAS MCs were recorded to confirm the conjugation reaction.

Preparation of FA-targeted RSV/MYP-loaded CAS MCsTo prepare FA–CAS MCs (F1), 10 mg RSV and 10 mg MYPs (5 mg MNS and 5 mg ANK) were dissolved in 95%ethanol and then added dropwise to 10 mg/ml aqueous FA–CAS solution (pH 7.4) under gentle magnetic stirringfor 24 h. The prepared FA–CAS MCs (F1) was then lyophilized using 2.5%w/v mannitol as a cryoprotectant for48 h in Cryodos-50 lyophilizer (Telstar Cryodos, Terrassa, Spain). The freeze-dried MCs were stored at 25◦C in adesiccator till further analysis [15,28].

Preparation of PEGPC–CAS MCsTo prepare MYPs-loaded CAS MCs, MYPs (5 mg MNS and 5 mg ANK) were dissolved in 95% ethanol andadded dropwise to aqueous CAS solution (2 mg/ml, pH 7.4) under gentle magnetic stirring for 24 h. PEGylatedphospholipid–RSV complex (PEGPC–CAS MCs, F2) was prepared via film-hydration method [29]. Briefly, a propermolar ratio (1:2) of RSV and phospholipids (Lipoid S75:DSPE–PEG 2000 [1:1 w/w]) was dissolved in 10 mlmixture of chloroform and methanol (1:1 v/v) and left overnight at room temperature with gentle stirring to allowthe formation of RSV-PEGPC complex. A thin drug–phospholipid complex layer was prepared by the removing oforganic solvents at a rotary evaporator (50 rpm and 45◦C). The flask was kept under vacuum for 1 h to ensure theevaporation of residual solvent. The PEG phospholipid–RSV complex was then used to envelop the MYP-loadedCAS MCs [30]. Thus, the thin film was hydrated for 1 h using the dispersion of previously prepared MYPs-loadedCAS MCs. The developed coloaded (PEGPC–CAS MCs, F2) was then lyophilized using 2.5% w/v mannitol for2 days and left in the desiccator [31]. The composition of (FA–CAS MCs, F1) and (PEGPC–CAS MCs, F2) isillustrated in Table 1.

Physicochemical characterization of the nanocarriersEntrapment efficiency (EE%) of both RSV and MYPs into CAS MCs was evaluated. An accurately weighed 20 mgof (FA–CAS MCs, F1) or (PEGPC–CAS MCs, F2) was mixed with 95% ethanol and metered to a constant volume

future science group 10.2217/nnm-2018-0006


under ultrasonication. Then, this solution was filtered using a membrane filter (0.45 μm), and then analyzed byHPLC. An HPLC method was developed for determination of RSV and MYPs simultaneously. An Agilent 1260Infinity HPLC system with a quaternary pump, an autosampler, diode array detector and an Agilent Chemstationdata processing system was used for the analysis. The HPLC analysis was carried out with an Agilent ZorbaxEclipse XDB C18 reversed-phase column (250 × 4.6 mm, 5 μm size of particle) maintained at room temperature.For chromatographic elution, the rate of flow was 1 ml/min over the entire separation and the injection volumewas 10 μl. A step gradient method was utilized for elution in which the mobile phase consisted initially of 50%acetonitrile and 50% acidified water (0.05% v/v acetic acid) for the first 5 min, followed by 80% acetonitrile and20% acidified water for the next 12 min. Total run time was 17 min. RSV was detected at 306 nm at tR of 4.2 min,while MYPs (both MNS and ANK) were detected at 386 nm (at tR of 11.64 and 13.67 min, respectively). Theencapsulation efficiency (%EE) and % drug loading (%DL) were calculated using the following equations:

% EETotal drug-Free drug

Total drug 100 (Equation 1)

% DLTotal drug-Free drug

Total micelles recovered 100 (Equation 2)

The particle size (PS), polydispersity index (PDI) and zeta potential profiles of the nanocarriers were determinedby dynamic light scattering (DLS) in a NanoZS/ZEN3600 Zetasizer (Malvern Instruments Ltd, Malvern, UK)with the sample previously diluted in ultrapure water. All of the DLS records were measured at 25◦C as tripletmeasurements. For the zeta potential measurement, nanocarrier suspension diluted in 1 mM KCl solution wasplaced in a universal folded capillary cell equipped with platinum electrodes. The zeta potential values wereestimated from the average electrophoretic mobility, as analyzed by Laser Doppler Anemometry.

The in vitro release of RSV and MYPs from both CAS MCs was determined using the dialysis membranemethod compared with free drug solution [32,33]. An aliquot (2 ml) of micellar suspension containing determineddrug amount (2 mg RSV, 1.2 mg MNS and 1.2 mg ANK) was transferred into dialysis bags (12–14 kDa MWCOVISKING dialysis tubing, SERVA, Germany). The bags were suspended in 200-ml phosphate-buffered saline(PBS), pH 7.4, with 0.2% w/v Tween 80, maintained in a water bath at (37 ± 0.5◦C, 100 rpm). After specificintervals, certain volume (2 ml) of the release medium containing the free drugs was transferred out and anotherequal volume of fresh release medium was added. All samples were run in triplicates and filtered using a syringefilter (0.45 μm), the released amount of RSV, MNS and ANK was analyzed by HPLC.

The physical stability of dual-loaded nanocarriers was evaluated according to time in both colloidal and solidstate. Therefore, the micellar suspension was kept at 4◦C in sealed tubes. PS, PDI and zeta potential were monitoredat several time points for 3 months. Moreover, the lyophilized dual drug-loaded MCs were re-evaluated for thoseparameters after 6-month storage in a desiccator at 25◦C.

Morphological analysisBoth types of CAS MCs were examined for their morphological characteristics using transmission electron mi-croscopy (TEM; JEM-100 CX, JEOL, Tokoyo, Japan). A 50-fold diluted dispersion of F1 and F2 in distilled waterwas mounted on the carbon grid. The dispersion was stained with uranyl acetate solution for 1 min [34]. The excessstain was removed with a filter paper, and the sample was left for air drying.

Redispersibility testingSuspension (2 ml) of both F1 and F2 containing mannitol (5% w/v) as cryoprotectant was transferred into5-ml glass vials and frozen at -80◦C, then lyophilized for 24 h. The lyophilized nanoparticles (NPs) were keptin a desiccator at 25◦C. For reconstitution, a certain weight of lyophilized MCs cake was mixed with 2 ml ofultrapure water followed by gentle agitation. The reconstituted NPs were analyzed for PS, PDI and zeta potential.Moreover, the redispersibility index (RI, the ratio between PS after and before lyophilization, respectively) and thelyophilization yield were calculated for all formulations.



In vitro hemolysis

A screening assay of hemolytic activity was evaluated by measuring hemoglobin release from erythrocyte afterincubation with (FA–CAS MCs, F1) and (PEGPC–CAS MCs, F2). Briefly, rat blood samples were collected intoethylenediaminetetraacetic acid containing test tubes, centrifuged and washed with saline. Red blood cells (RBCs)suspension was prepared by preparing tenfold dilution of RBCs with saline (1:9 v/v). Then, the RBCs suspension(2 ml) was incubated for 1 h with an equal volume of the micellar dispersion using two concentrations for eachnanosystem (100 and 250 μg/ml), at 37◦C with gentle shaking. After sample centrifugation (3000 rpm for 5 min),the supernatant absorbance (A) was measured at 545 nm using UV–Vis spectrophotometry. Both negative andpositive control solutions were prepared by addition 2-ml RBC suspension to the same volume of saline (0% lysis)or 1% Triton X100 (100% lysis) for negative and positive controls, respectively. The hemolytic rates of the sampleswere calculated according to the following equation [35].

Hemolytic rate A A

A A t nc

pc nc

(%)( )

( )

100 (Equation 3)

At, Anc and Apc: absorbance value of test sample, negative and positive controls, respectively.

In vitro cytotoxicity study

This study was achieved using human breast adenocarcinoma MCF-7 cell line (purchased from American TypeCulture Collection, ATCC, USA). The cells were kept in Dulbecco’s modified Eagle medium (DMEM) containing10% fetal bovine serum in a CO2 incubator (5% CO2 at 37◦C). Cells were seeded at a density of 5 × 103/well ina 96-well plate containing 100 μl of DMEM and allowed to adhere to the plate overnight. The cytotoxicity of thefree drugs and drug-loaded CAS MCs (F1 and F2) on MCF-7 cells was assessed by the MTT assay [36]. Therefore,the medium was substituted by fresh medium containing various concentrations of the drugs either as solution orencapsulated in MCs and incubated for another 24 h. Then, 100 μl of MTT solution (0.5 mg/ml in DMEM)was added to replace the culture medium and incubated for further 4 h at 37◦C under the dark. The supernatantwas removed by centrifugation at 2000 rpm for 10 min, and then the elaborated MTT-formazan crystals weredissolved by addition of 100 μl of DMSO to the wells and maintained for 15 min under agitation. Absorbanceof the converted dye was recorded at a wavelength of 570 nm using a microplate reader (Model 550, Bio-Rad,CA, USA). The cell viability was expressed as percentage of the untreated control wells. The IC50 values weredetermined using Origin 8.0 (Origin Lab, MA, USA) depending on the fitted data.

In vivo studyAnimals & developing of tumor model

In this study, female BALB/C mice (7–8 weeks, 25 ± 5 g) were housed in stainless-steel mesh cages, under standardtemperature, illumination, humidity, food and water supply. Female BALB/c mice were subcutaneously inoculatedinto the left side of the mammary fat pad with approximately 107 of EAT cells suspended in PBS (National Instituteof Cancer, Egypt). EAT cells were obtained from the ascitic fluid of inoculated BALB/c mice with 8–10 days oldascitic tumor [37]. Tumor growth was checked daily until its volume reached 100 mm3. Tumor volume was assessedbased on tumor’s perpendicular diameters using the following equation:

Tumor volume / JI minor axis X major axis ( ) ( )4 3 2(Equation 4)

In vivo antitumor efficacyAnimals were categorized into eight groups (eight mice/group). These groups included positive control (untreatedEAT-inoculated mice), negative control (healthy mice receiving saline), blank FA–CAS MCs, free MYPs, free RSV,free combined MYPs-RSV cosolvent composed of DMSO:PEG400:saline (0.5:3.5:6 v/v/v), FA–CAS MCs, F1and PEGPC–CAS MCs, F2-treated groups. The tumor-bearing mice were injected in the tail vein with the freedrugs or MCs equivalent to 5 mg/kg per day of RSV, for consecutive 3 weeks. Each mouse was tail-markedand followed individually throughout the whole experiment. To avoid the hypoglycemic effect of the MYPs, allgroups received 1 ml of 10% w/v dextrose by oral route prior to each treatment dose. All the surviving mice were



sacrificed after 21 days by cervical dislocation. The excised tumors were divided into two parts; one part was usedfor assessment of tumor biomarkers and the second part was used for histopathological studies. The tumor volume,the weight of the tumors after excision and percentage body weight change for all groups throughout the treatmentcourse were determined.

Tumor growth biomarkers

Homogenization of the excised tumors in cold PBS was performed, a final 40% tumor homogenate was divided intofive fractions for quantitative determination of tumor biomarkers. Aromatase expression level was quantified using‘AROBioAssay ELISA Kit’ (US Biological Life Sciences, USA). Angiogenesis was expressed in the term of VEGFlevel using ‘VEGF ELISA Kit’ (RayBio Tech Inc., USA) and Cyclin D1 (Rat Cyclin-D1 ELISA Kit, EIAab™).Apoptosis was determined according to the level of tissue caspase 3 using ‘Caspase 3 (Casp-3) ELISA Kit’ (WKEAMed Supplies Co., USA). NF-κB was determined using ‘Bio Source NF-κB ELISA kit’ (BioSource InternationalInc., USA). All the markers were quantified based on the manufacturing protocol.

Histopathological studyPreservation and fixation of the tumor samples were performed using neutral formalin (10%) for 24 h. Tissuesections (5 μm thick) were immersed in distilled water, stained with hematoxylin for 5 min and eosin for 2 min,dehydrated in alcohol and mounted in Canada balsam prior to examination by microscope. Necrosis scores areprovided in the Supplementary Materials section.

Statistical analysisIBM SPSS software package (version 20) was used to analyze the in vivo results. Analysis of Variance test and Tukey’sMultiple Comparison test were utilized for pair-wise statistical comparisons between the investigated groups. Resultssignificance was judged at the 5% level.

Results & discussionPhysicochemical characterization of prepared MCsAs an active tumor-targeting approach, FA was coupled to CAS via simple carbodimide formation. The carboxylicgroup of FA was activated in the presence of DIC and Oxyma into active ester intermediate. This intermediate hasreduced racemization level and suppresses the undesirable side reactions [38]. For further structural investigations,UV spectra of FA, CAS MCs and FA–CAS MCs conjugates were recorded. As shown in Figure 1, FA spectrumshowed its characteristic peaks at 258, 285 and 365 nm. On the other hand, spectrum of CAS MCs exhibitedonly the characteristic 280-nm protein peak with no peaks in the 360-nm region. Interestingly, the spectrum ofour developed FA–CAS MCs showed peaks at 356, 288 and 367 nm corresponding to the folate moiety. Thisconfirmed the chemical conjugation of folate to CAS elaborating FA–CAS conjugate [39].

The lyophilized FA–CAS conjugate was obtained as deep yellow powder with conjugation efficiency of 97% cal-culated by the calibration curve method. Depending on intrinsic amphiphilic characteristic of CAS, the hydrophobicdrugs could be entrapped within CAS hydrophobic interior. Consequently, in our work, both hydrophobic MYPsand RSV were encapsulated within the lipophilic cargo of FA–CAS MCs. Blank FA–CAS MCs investigated a PSof (124 ± 3.0 nm), which increased to 137 ± 5.0 nm after RSV + MYPs dual loading (Table 1 & Figure 2A).The PS increment confirmed successful drug encapsulation. FA–CAS MCs, F1 exhibited negative zeta potential(-21 ± 1.9 mV) reflecting a good colloidal stability (Table 1). These results are in accordance with Teng et al.’s find-ing, who synthesized curcumin-encapsulated FA-conjugated soy protein nanoparticles with zeta potential rangedfrom -36 to -42 mV [40]. RSV, MNS and ANK were efficiently encapsulated into FA–CAS MCs with %EE of 86.0,86.3 and 85.82% w/w, respectively.

In another approach, coating of CAS MCs with PEGylated phytosomal RSV complex layer produced a compactmultireservoir nanosystem that allows passive accumulation in the tumorous site via EPR mechanism. SuccessfulRSV complex formation was confirmed by disappearance of its melting endothermic peak in the complex thermo-gram (mentioned in Supplementary material section). The hydrophilic interaction between Î–CAS-enriched surfaceand polar molecules of the phospholipid bilayer represented a stabilizing factor for this nanovehicle. PEGPC–CASMCs, F2 exhibited larger PS (272.6 ± 7.0 nm) in relative to (FA–CAS MCs, F1), (Figure 2A), with a PDI of0.21 ± 0.03 and negative zeta potential (-36.8 ± 2.1 mV), (Figure 2B). Similar observation were reported byHe et al. where PEGylated mesoporous silica of different PSs (80–360 nm) were prepared, however, PEGylated



220 270 320

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Ab

so

rban

ce

370 4200.0

0.5

1.0

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Figure 1. UV–VIS spectra of folic acid, casein micelles and folate-conjugated casein micelles.

mesoporous silica with larger PS of 360 nm have been exerted extraordinarily stability and protection against beingcaptured in the spleen [41].

Compared with FA–CAS MCs, (PEGPC–CAS MCs, F2) demonstrated higher %EE of RSV (99.0%) against86.0% for (FA–CAS MCs, F1). This could be explained by the highly immersion of RSV in the lipidic bilayeraugmented by successful complex formation of RSV with phospholipids via both hydrogen bond and van der Waalsbond. Similar entrapment of genistein (97.9%) within the phospholipid bilayer enveloping poly(lactic-co-glycolic)(PLGA)-paclitaxel NCs was reported [30]. Both lipophilic MNS and ANK were highly encapsulated in both typesof MCs may be due to their high affinity to the hydrophobic micellar core.

Under TEM, both FA–CAS MCs and PEGPC–CAS MCs were demonstrated uniform size and smooth surfacewith no aggregation confirming high colloidal stability. PS reported by TEM was smaller than that reported byDLS (108.2 ± 11.10 and 129.97 ± 9.1 nm for FA–CAS MCs [F1] and PEGPC–CAS [F2], respectively) dueto particle shrinkage during the drying step in TEM analysis instead of the presence of the hydrodynamic layeraround the MCs in the colloidal form [33]. Interestingly, the core–shell-type structure of PEGPC–CAS MCs wasdemonstrated with the PEGylated lipid bilayer around 24.76 nm (Figure 2C & D).

In vitro releaseCommonly, in all drug-release experiments, the release pattern of drugs from the developed nanoformulationswas compared with free drug solution to detect the capacity of the nanocarriers to provide sustained drug releasecompared with fast release from solution. This sustained release profile is required to prevent the prematuredrug release into systemic circulation and keep it released at tumor sites. Both folate-conjugated and PEGylatedCAS MCs exhibited a biphasic-release pattern of RSV, MNS and ANK showing initial burst release followed bysustained released profile (Figure 3A & B). After 4 h, about 43.6% of RSV was released from PEGPC–CAS MCscompared with only 12.78% from FA–CAS MCs. This can be explained by influence of both PEGylation [42] andphospholipid complex formation on RSV solubility and release behavior. Bioavailability and clinical applicationof another botanical polyphenols (Curcumin, Silymarin and Green Tea Extracts) were improved substantially inthe form of phytosomes via phospholipids-complex formation [43]. After 24 h, about 89.3 and 98.8% of RSV was



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Record 4: FACAS MCs (F1)

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Figure 2. Physicochemical characterization of the prepared casein micelles. (A) Particle size distribution diagrams of drug-loaded singlecompartment (FA–CAS MCs, F1) and multireservoir PEGylated phospholipid bilayer-enveloping CAS MCs (PEGPC–CAS MCs, F2), (B) Zetapotential diagrams of (PEGPC–CAS MCs, F2), (C) TEM micrograph showing morphology of (FA–CAS MCs, F1) and (D) TEM image of(PEGPC–CAS MCs, F2) with core–shell structure.CAS MC: Casein micelle; FA–CAS MC: Folate-conjugated casein micelle; PEGPC–CAS MC: PEGylated RSV-phospholipid complex bilayerenveloping casein-loaded micelle; RSV: Resveratrol; TEM: transmission electron microscopy.

released from FA–CAS MCs (F1) and PEGPC–CAS MCs (F2), respectively. In comparison, 100% free RSV wasreleased from its solution within 2 h.

Regarding the release of MNS, the PEGylated phospholipid bilayer was observed to reduce its burst releaseafter 4 h from 49.9% for FA–CAS MCs (F1) to 28.5% for PEGPC–CAS (F2) followed by a slow release of theremaining drug up to 90.0 and 73.7%, respectively, after 24 h. MNS released from the micellar hydrophobic corehas to diffuse through an extra barrier, as phospholipid bilayer is acting as a molecular fence that leads to retainthe drug molecules in the internal core. Controlled and sustained drug-release profiles of docetaxel was observedfrom PLGA–lecithin–PEG NPs where the degree of lipid covering influenced its drug release [44]. With the samemanner, similar release behavior was previously reported for letrozole where PEGylated protamine NCs effectivelyreduced the initial burst release of LTZ release after 4 h compared with PRM-NCs and HA-NCs [22].

A more powerful prolonged release pattern of ANK was reported from both folate and PEGylated micellarsystems. This behavior may be attributed to the higher hydrophobicity of ANK compared with MNS resulting inhigher affinity to the hydrophobic cargo of MCs. Therefore, about 70.6 and 60.6% of ANK was released fromFA–CAS MCs (F1) and PEGPC–CAS (F2) after 24 h, respectively.



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Physical stability & redispersibilityThe storage stability of our prepared nanoformulae in both colloidal and solid state was evaluated. FA–CAS MCs,F1 demonstrated a gradual PS increase when stored in colloidal form at 4◦C (140.1 ± 2.1 and 186.2 ± 1.3 nmafter 2 and 4 weeks, respectively) compared with initial size (137.9 ± 3.0 nm). However, after 3 months, FA–CASMCs showed a dramatic size increase reaching 505 ± 4.1 nm, this observation may be result of origination of minoralterations in protein secondary structure as the FA–CAS interaction might affected the MCs colloidal stability onthe long run [45]. Regarding to surface charge, approximately no obvious change in zeta potential compared withinitial measurement (-25 ± 2.0 mV). In parallel, the physical stability of CAS MCs was improved via RSV-PEGPCbilayer enveloping. After 3 months of PEGPC–CAS MCs (F2) colloidal storage, mean PS became slightly larger inrelative to the initial measurements (300 ± 5.0 nm after 3 months) versus (270 ± 10.63 nm starting measurement).Moreover, no change in zeta potential was observed, see Figure 3C. Generally, due to steric stabilization by thePEGylated shell, increased stability may have been envisaged as reported for other PEGylated particle types [22].

Potential pharmaceutical applicability of these NPs needs scalable processes for the storage of larger batches ofNPs. However, the nanosystems were evaluated according to their solid-state stability using freeze-dried powderedform, mannitol (2.5% w/v) was used to enhance the drying and redispersibility of lyophilized nanocarriers. These



Table 2. Effect of freeze-drying on the redispersibility and physicochemical characteristics of dual drug-loaded caseinmicelles.

Particle size (nm) PDI RI (Sf/Si) Zeta-potential (mv) Yield (%w/w)

Before After Before After Before After

FA–CAS MCs (F1) 145 ± 8.0 140 ± 6.2 0.229 ± 0.2 0.265 ± 0.2 0.966 -21.0 ± 1.0 -19.0 ± 1.5 95.71 ± 2.0

PEGPC–CAS MCs(F2)

260 ± 10.0 270.4 ± 4 0.239 ± 0.11 0.23 ± 0.02 1.038 -29.5 ± 4.1 -29.0 ± 3.2 83.73 ± 5.1

FA–CAS MC: Folate-conjugated casein micelle; PDI: Polydispersity index; PEGPC–CAS MC: PEGylated RSV-phospholipid complex bilayer enveloping casein-loaded micelle; RSV: Resveratrol;RI: Redispersibility index (final particle size/initial particle size).

additives are commonly added in order to protect the nanoparticles from the stress of freezing (cryoprotectant) ordrying (lyo-protectant) [46]. The lyophilized form of both prepared nanosystems investigated excellent stability withno change in PS (139 ± 5.1 and 265.2 ± 7.3 nm for FA–CAS MCs, F1 and PEGPC–CAS, F2, respectively) after6 months storage at room temperature in the desiccator compared with the initial (137 ± 5.0 and 272.6 ± 7.0 nmfor FA–CAS MCs, F1 and PEGPC–CAS, F2, respectively). Both formulae were freely redispersible in aqueousmedium as mannitol allows for recovery of (140 ± 6.2 and 270.4 ± 4.0 nm for FA–CAS MCs, F1 and PEGPC–CAS, F2 respectively), similar to the original measurement (145 ± 8.0 and 260 ± 10.0 nm) that is showing anacceptable range of RI (Sf/Si ratio) values of 0.966 and 1.038 for (FA–CAS MCs, F1) and (PEGPC–CAS MCs,F2), respectively, Table 2. Therefore, storage of MCs in a lyophilized form rather than a liquid dispersion wasfound to be an effective to induce the stability of MCs and preserve their size characteristics. Similar observationwas reported when PLGA–lecithin–PEG NPs were prepared in freeze dried form using (10% w/v) sucrose as acryoprotectant and allows for recovery of 87.0 ± 0.6 nm NPs versus the original 64.5 ± 0.5 nm diameters [44].

In vitro hemolysis testHemolysis in vivo can cause anemia, jaundice and other pathological symptoms, so acceptable hemolytic activityof NPs (<5%) represented a crucial factor for any parenteral delivery system [47]. Two concentrations of eachnanosystem were evaluated in this study (100 and 250 μg/ml). At a concentration of 250 μg/ml, the percentagehemolysis induced by FA–CAS MCs, F1 and PEGPC–CAS MCs, F2 was 1.3 and 1.8%, respectively. At lowerconcentration of 100 μg/ml, the percentage hemolysis of folate-targeted MCs was obviously decreased to 0.22%while the PEGylated nanosystem still had approximately the same value (1.7%), see Figure 4A & B. The lowerhemolysis activity of folate nanosystem compared with PEGylated one may be attributed to the protective effectof highly hydrated k-CAS hairs, which act as a protective coat on the surface of CAS MCs along with folatemoieties that hinder their interaction with RBCs. Furthermore, the negativity of our nanosystems’ surface possessless damaging effect on the negatively charged RBCs outer membrane due to the repulsion effect and hencelonger blood circulation and lower opsonization [48]. Our results are in agreement with the finding of Li et al.,who reported the hemocompatibility of mitomycin-soybean phosphatidylcholine complex-loaded PEG lipid-PLAhybrid nanoparticles with folate functionalization where no significant hemolytic effects were observed even at theelevated concentration [49]. Similarly, it should be kept in mind that PEG steric barrier was conceived with theidea of preventing the absorption of lysozymes and reducing the interaction with RBCs. Zhu et al. suggested thatPEGylation of chitosan NPs could be a successful approach for reduction of NPs hemolytic activity [50].

In vitro cytotoxicityClearly, both developed MCs (F1 and F2) were significantly effective in inhibiting growth of MCF-7 cells at theconcentration range of (0–50 μg/ml). In relative to free drug combination, RSV + MYPs (IC50 = 37.9 μg/ml),FA–CAS MCs, F1 and PEGPC–CAS MCs, F2 showed IC50 of 20.3 and 20.5 μg/ml, respectively, after 24 h,reflecting their superior antineoplastic efficiency (p < 0.05*), (Figure 4C & D). At higher concentration (50μg/ml)of the drug, there was no change in percentage survival index for F2 and free RSV + MYPs, which may be due tothe effect of release pattern and the difference in cellular uptake mechanisms.

Blank nanocarriers demonstrated very little toxicity to MCF-7 cells due to the change of the tumorous environ-ment by EPR (viability was >80% after 24 h). The inhibition of MCF-7 cells by FA–CAS MCs, F1 can be ascribedto rapid incorporation into MCF-7 cells via the specific ligand–receptor (folate–folate receptor) interaction. Thisresult is in agreement with the previously reported higher uptake of covalent-conjugated FA-HSA NPs into cancercells [51]. Recently, Hao et al. reported the superior cytotoxicity of folate-targeted RSV-loaded Pluronic/TPGS-



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mixed MCs (IC50 = 27.89 μg/ml) against folate receptor-overexpressing MCF-7 cells compared with free RSV(137.58 μg/ml) after 48 h [52]. Furthermore, the structure resemblance of phospholipid bilayer to the lipid contentof the mammalian cellular membrane can increase membrane penetration. In our laboratory, PEGylated protamineNCs enhanced the cytotoxicity potency of LTZ-CXB combination compared with free combined drug solutionagainst MCF-7 (IC50 = 14.4 μg/ml) [22].

In vivo assayTumor volume & weight

In this part, each group was treated with FA–CAS MCs, F1 and PEGPC–CAS MCs, F2 in PBS at the dose of5 mg/kg per day up to 3 weeks along with free drugs. Compared with FA–CAS MCs, F1 and PEGPC–CAS, F2the positive control, free drugs and blank nanosystem groups demonstrated progressively tumor volume increasethroughout the period of the treatment. The set of positive control had demonstrated more pronounced anduncontrollable increase of tumor volume corresponding to 1466.3 mm3, which was higher than that observed after



treatment with free RSV (890.6 mm3) and free MYPs (1030.1 mm3). The tumor volume of blank FA–CAS MCsgroup showed no significant difference compared with positive control. Coadministration of free RSV + MYPslimiting the increase of tumor volume to (770.55 mm3) during the same course of treatment regimen indicatedgood combination selection. Interestingly, antineoplastic activity was enhanced via coincorporation of our novelcocktail into the micellar systems [15]. At the end of the treatment course, PEGylated MCs were superior tofolate-conjugated MCs as evidenced by higher tumor regression (694.3 and 590.3 mm3) for FA–CAS MCs, F1and PEGPC–CAS, F2, respectively, Figure 5A.

The weight of the excited tumors was also determined for each group. PEGylated- and folate-treated groupsdemonstrated obvious reduction in average tumor weight compared with other groups ensuring excellent antineoplastic effect of our nanosystems (data not shown). The nanosize, folate-conjugation and PEG coating helpenhancing the permeation of CAS MCs across the cellular membrane. Geng et al. demonstrated the best tumorgrowth suppression effect of RSV-loaded RGD-conjugated HSA nanoparticles via a PEG bridge by virtue of theimproved RSV biocompatibility and prolonged circulation [53].

Although RSV seems to have a negligible toxicity limit [54], the antidiabetic effect of both RSV [55] and MYPs [56]

can explain the higher percentage body weight loss in free drugs combination-treated groups (16.00%) recordedat the end of the study in relative to coloaded micellar systems. Interestingly, FA–CAS MCs, F1-treated groupdemonstrated reduced body weight loss to 5.04%, while PEGPC–CAS MCs (F2)-treated group showed an increaserather than decrease in body weight by 2.7% confirming the efficiency of the PEGylation strategy in toxicityreduction (Figure 5B). This result may be attributed to the elevated systemic toxicity of chemotherapeutic drugs intheir free state, while utilizing nanovehicle as homing devices for these drugs could likely protected the mice fromtheir toxic effects.

Determination of tumor growth biomarkers

Our selection of naturally chemosensitizers of RSV and MYPs could exert synergistic effects on aromatase levelinhibition in cancer cells as RSV is a potential inhibitor in the aromatase synthesis pathway at the enzymeand mRNA levels [57]. In addition, docking studies presented the most constructive binding mode of MNSand ANK to aromatase exposed the capability of these natural biopigments to treat breast cancer via aromatasetargeting [10]. Regarding the aromatase level, a comparable pattern of response was observed where all treated groupsdemonstrated lower aromatase levels in comparison to the untreated positive control, which showed an aromataselevel of 77.14 ng/mg tissue protein. The significant aromatase level reduction (p < 0.05*) was recorded for both(FA–CAS MCs, F1) and (PEGPC–CAS MCs, F2) treated groups corresponding to 6.14 and 7.89 ng/mg tissueprotein, respectively (Figure 5C). The superiority of the coloaded nanovehicles over the free RSV + MYPs cosolventcould be directly related to the enhanced cellular permeation via both PEGylation and folate receptor targetingof dual-loaded nanostructured delivery system reflected on the reduction of tumor development and aromataseinhibition with subsequent reduction of de novo estrogen synthesis.

VEGF is overexpressed in almost all cancer types, however, no VEGF expression in normal tissues. Althoughthere was no direct information which explains the effect of MYPs on tumor angiogenesis, we supposed a positiverelationship between MYPs anti-inflammatory effect and cancer metastasis control. Both folate and PEGylatedmicellar systems were significantly (p < 0.05*) reduced VEGF level in comparison with the positive control andfree drugs-treated groups (6.44 and 9.95 ng/mg tissue protein, for (FA–CAS MCs, F1) and (PEGPC–CAS MCs,F2), respectively (Figure 5D). That is because specific receptor-mediated interaction enhances NPs uptake into thetargeted cells.

Cyclin D1 is a marker of proliferation that regulates the cell cycle essentially in G1 phase. It is a candidateproto oncogene contributed in pathogenesis of several human tumors with high-expression level, especially inbreast cancer. With the same manner, both folate and PEGylated formulae were significantly (p < 0.05*) reducedcyclin D1 level by 5.4- and 4.94-folds, respectively, when compared with free drug combination-treated groups(Figure 6A).

NF-κB has turned out to be a substantial therapeutic target in cancer remedy as it shown to be activated incancer tissues. Incorrect regulation of NF-κB has been linked to carcinogenesis, inflammatory and autoimmunediseases. These investigated measurements could confirm the capacity of our novel fabricated nanovehicle to exerta significant reduction effect on NF-κB level (p < 0.05*). All treated groups exhibited a significant lower NF-κBlevel compared with positive control and free drug cosolvent confirming our great success in the selection of this



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Figure 5. In vivo anti-tumor efficacy. In vivo antitumor efficacy demonstrating (A) average tumor volume (mm3) measured throughoutthe study period, with corresponding excited tumor images showing the differences between mice groups at the end of the treatmentcourse. (B) Percent change of body weight of EAT mice treated with free drugs combination compared with dual-loaded (FA–CAS MCs,F1) and (PEGPC–CAS MCs, F2) for 3 weeks compared with untreated positive control. (C) The level of aromatase and (D) VEGF tumorbiomarkers measurement in tumor homogenate of EAT animal treated with free drugs combination and dual drug-loaded (FA–CAS MCs,F1) and (PEGPC–CAS MCs, F2) compared with untreated positive control (a: p < 0.05 vs positive control, d: p < 0.05 vs RSV + MYP, f: p< 0.05 vs [FA–CAS MCs, F1], h: p < 0.05 vs [PEGPC–CAS MCs, F2]).EAT: ; FA–CAS MC: Folate-conjugated casein micelle; MYP: Monascus yellow pigment; PEGPC–CAS MC: PEGylated RSV-phospholipidcomplex bilayer enveloping casein-loaded micelle; RSV: Resveratrol.

novel drugs cocktail (p < 0.05*). FA–CAS MCs (F1) and PEGPC–CAS (F2) highly reduced NF-κB level by 8.22-and 5.68-folds, respectively, when compared with free RSV + MYPs cosolvent-treated group (Figure 6B).

In addition, the apoptotic rate in mice tumor tissue was evaluated by measuring caspase-3 level as a markerof apoptosis. It was reported that both RSV and MYPs could induce cellular apoptosis through various points inthe apoptotic pathway. In our work, all treated groups displayed elevated apoptotic effect with a significant higher



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Figure 6. Tumor growth biomarkers & histopathological analysis. The level of tumor biomarkers measurement in solid tumorhomogenate including (A) CD1, (B) NF-kB, (C) apoptotic marker caspase-3 and (D) histopathological H&E analysis showing necrotic score(scoring system of necrosis from 1 to 4) of EAT mice treated with free drug combination and dual drug-loaded (FA–CAS MCs, F1) and(PEGPC–CAS MCs, F2) compared with untreated positive control. Score 4: section in poorly differentiated tumor showing >50% necrosis;Score 3: section in poorly differentiated tumor showing about 35% necrosis; Score 2: section in poorly differentiated tumor showingabout 25% necrosis; Score 1: section in poorly differentiated tumor showing about 10% necrosis. The mean value of all ten scores wascomputed for each excised tumor and expressed as mean of necrosis scale ± standard error (a: p < 0.05 vs positive control, d: p < 0.05 vsRSV + MYP, f: p < 0.05 vs [FA–CAS MCs, F1], h: p < 0.05 vs (PEGPC–CAS MCs, F2]).FA–CAS MC: Folate-conjugated casein micelle; H&E: Hematoxylin and eosin; MYP: Monascus yellow pigment; PEGPC–CAS MC:PEGylated RSV-phospholipid complex bilayer enveloping casein-loaded micelle; RSV: Resveratrol.



caspase-3 level (p < 0.05*) compared with untreated positive control group. FA–CAS MCs (F1) and PEGPC–CAS(F2) increased caspase-3 level by 3.59- and 2.9-folds, respectively, when compared with free RSV + MYPs-treatedgroup (Figure 6C).

From these outcomes, both FA–CAS MCs (F1) and PEGPC–CAS MCs (F2) treated groups demonstrateda significant reduction (p < 0.05*) for all measured tumor biomarkers level compared with untreated positivecontrol and free drugs combination-treated group. Upon comparing the total antitumor efficacy of FA–CAS MCs(F1) and PEGPC–CAS MCs (F2) to each other, we excluded that although (FA–CAS MCs, F1) exerted slightly(nonsignificant) higher effect on reducing the tumor growth biomarkers levels compared with PEGPC–CAS MCs(F2), a slight increase in the average tumor volume of F1-treated group (694.3 mm3) was recorded after 3 weekscompared with that measurement of F2-treated group (590.3 mm3). This observation could be explained as aresult of great necrotic damage of cancer cells and neighboring tissues that stimulate an inflammatory response forhealing [58].

Histopathological study

Additional evidence of the induced anticancer efficiency of the RSV-MNS-ANK-loaded MCs was analyzed byhistological examination. The abundant tumor cells were investigated in the positive control group. On thecontrary, the extensive dead cells without nuclei were reported in the tumor treated with FA–CAS MCs (F1) andPEGPC–CAS MCs (F2) as both yielded greater capacity in inducing the cell apoptosis/death and reducing thecell proliferation, which was explained by higher scoring of necrosis (2.64 ± 0.05 and 2.96 ± 0.07, respectively)compared with free combination-treated groups (2.08 ± 0.03%). Our investigation can be explained by the abilityof negatively charged nanodelivery system for cellular internalization, which is believed to occur through nonspecificaccumulation of MCs on the plasma membrane’s cationic sites [59]. Moreover, the folate targeting and PEGylatedproperties greatly improved the therapeutic activity of the loaded NPs because of the higher clustering at the tumorsite and the greater cellular uptake by the tumor cells (Figure 6D). Our results are in agreement to the findings ofMartinez et al., who showed the most quiescent and disorganized structures of breast cancer xenograft tumors aftertreatment of tamoxifen-loaded folate-targeted BSA nanoparticles [60].

According to the abovementioned results, there is a clear evidence on the enhancement of antineoplastic activityof RSV in multiple-step strategy including: Coadministration of monascus biopigments with RSV can enhance theantitumor efficacy of both drugs through several mechanistic pathways as confirmed by synergistic reductionof tumor biomarkers. Dual encapsulation of RSV and MYPs combination into CAS micellar nanosystems canprovide full protection of both drugs, sustainable drug release and improved cell membrane penetration potency.Preparation of novel folate-targeted coloaded RSV/MYPs CAS MCs in this step dual utility of both ultrasmall size andligand-receptor binding of nanodelivery system were achieved. In addition to nanosize-mediated EPR effect, folate-receptor targetability has been exploited since FA receptors are highly abundant on the surface of metabolicallyactive breast cancer cells, resulting in additional specific cellular internalization and extra-antineoplastic activity ofboth. Envelopment of MYPs-loaded CAS MCs within PEGylated phytosomal RSV bilayer to evade opsonization thusavoiding reticuloendothelial system uptake leading to prolonged systemic circulation as well as enhanced releaseof RSV via complexation with phospholipids. The improved colloidal stabilization of CAS MCs via steric andelectrostatic effects, and the enhanced permeation through cancer cell membranes may be both mediated by theunique phosphatidylcholine composition.

ConclusionA novel combination rationale of RSV + MYPs was selected based on their basic mechanistic pathways. We fabricatedtwo novel nanodelivery systems including RSV-MYPs dual-loaded FA–CAS MCs to enable tumor targeting, inparallel with PEGPC–CAS MCs as passively nanoparadigm for breast cancer therapy. Both micellar nanosystemspresented an appropriate PS and zeta potential, a good solid-state stability, high EE and controlled drug release,which led to improved drug efficiency and reduced toxicity in the experimental animals. Both nanosystems exhibitedhighly selective tumor accumulation and greatly superior therapeutic efficiency in vivo confirmed by their potentialeffect on tumor volume, tumor growth biomarkers including aromatase, VEGF, CD-1, NF-κB, Caspase-3 as wellas histopathological studies. Both FA–CAS MCs and PEGPC–CAS MCs had comparable antineoplastic activity.Therefore, we suggested that both were robust and attractive drug delivery systems for effective chemotherapyagainst breast cancer.



Summary points

• A novel combination of herbal and microbial-derived antineoplastic drugs (resveratrol [RSV]/monascus yellowpigments [MYPs]) was selected based on their mechanistic pathways for breast cancer therapy.

• Active-targeted casein micelles (CAS MCs) were developed by carbodiimide coupling of folate to enhance theirinternalization into breast cancer cells via receptor-mediated endocytosis.

• Passively targeted multicompartmental nanocarriers were developed by enveloping MYPs-loaded CAS MCswithin PEGylated phospholipid-RSV complex bilayer prepared via thin film hydration.

• Both micellar types exhibited acceptable physicochemical properties, good stability profile and superiorcytotoxicity to breast cancer cells compared with free MYPs/RSV combination, which can be attributed toprolonged circulation, enhanced cellular internalization resulting in higher drug accumulation and improvedantitumor efficacy.

• Both MCs demonstrated powerful and comparable anticancer activity in Ehrlich Ascitis mammary tumor micemodel revealed as significant reduction of tumor volume and weight, suppression of aromatase, VEGF, CD-1,NF-κB and elevation of Caspase-3, augmented by histopathological studies.

• The dual drug-loaded micellar systems demonstrated reduced body weight loss of treated animals comparedwith the higher body weight loss in free drug combination-treated groups, thus confirming the ability of MCs toreduce the drug toxicity.

• Both folate-targeted CAS MCs and PEGylated RSV-phospholipid complex bilayer enveloping CAS MCs weresupposed to be potential nanodelivery systems for targeted breast cancer therapy.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or finan-

cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria,

stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined

in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human

subjects, informed consent has been obtained from the participants involved.

Supplementary data

To view the supplementary data that accompany this paper please visit the journal website at:

www.futuremedicine.com/doi/full/10.2217/nnm-2018-0006

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