Development of core-shell nanocarrier system for

European Journal of Pharmaceutical Sciences 118 (2018) 103–112

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European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps

Development of core-shell nanocarrier system for augmenting piperine cytotoxic activity against human brain cancer cell line Abanoub S. Sedekya, Islam A. Khalila,b, Amr Hefnawya, Ibrahim M. El-Sherbinya,

T

⁎

a

Nanomedicine Lab, Center of Materials Science (CMS), Zewail City of Science and Technology, 6th of October, Giza 12578, Egypt Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy and Drug Manufacturing, Misr University of Science and Technology (MUST), 6th of October, Giza 12566, Egypt b

A R T I C LE I N FO

A B S T R A C T

Keywords: Core-shell nanoparticles Micellization Trimethyl chitosan Piperine Human brain cancer cell

Brain tumor has a low prognosis with only 15% survival rate (5 years after diagnosis). Many of the current therapeutics have limited activity due to their inability to cross the blood brain barrier which retards drug accumulation in tumor site and causes drug resistance. Piperine, a phytochemical drug with poor solubility, could be an alternative to current therapeutics after evading its solubility and permeability limitations. Piperine micellization was optimized to improve drug solubility. Positively charged trimethyl-chitosan was synthesized then electrostatically adsorbed onto piperine nanomicelles forming core-shell nanoparticles. Physicochemical and morphological characterizations, and in-vitro release were performed. Cytotoxicity on human brain cancer cell line (Hs683) was evaluated using IC50 determination, cell cycle arrest analysis, apoptosis and enzyme-linked immunosorbent assay. Optimum piperine-loaded core-shell nanoparticles were successfully fabricated with double-phase release model. Significant improvement in cytotoxicity than free drug was noted with increasing in G2/M-phase and pre-GI-phase population, apoptotic/necrotic rates and inhibition of CDK2a.

1. Introduction Cancer is ranked as the leading cause of death in the developed countries and the second leading cause of death in economically developing countries (Jemal et al., 1999). Brain tumor patients are around 3.5 per 100,000 people where about 650 people are diagnosed with a malignant brain tumor daily as reported by Ferlay et al. (2010). However, the main cause of brain tumor is still under investigation (Herholz et al., 2012; Kadam, 2013). Over the last few decades, phytomedicines demonstrated a pivotal role in drug discovery where 50% of FDA approved drugs are of natural origin (Newman and Cragg, 2012). Black pepper (Piper Nigrum L.) is a perennial vine grown for its berries, and it is usually used as a spice and medicine. Piperine (PIP) is the major alkaloid in Piper Nigrum L. which has a wide range of biological activities including, for instance, being anti-depressant, bioenhancer, antioxidant, apoptosis inhibitor, anti-inflammatory, antihypertensive and particularly anti-tumor, where PIP suppresses tumor growth and metastasis (Elnaggar et al., 2015a, 2015b; Lai et al., 2012). However, the therapeutic applications of PIP are limited because of its immunotoxicity, poor aqueous solubility, and high first pass metabolism.

To overcome such limitations, some reported preliminary studies directed the efforts towards the use of nanotechnology (Elnaggar et al., 2015a, 2015b; Tyagi et al., 2011; Yusuf et al., 2013). These nanotechnology-based systems have been employed to overcome the main brain delivery limitations, particularly the blood brain barrier (BBB)hindered penetration taking into consideration the effect of both particle size and surface charge. For instance, nanoparticles (NPs) with high zeta potential (high positive charge) have shown a toxic effect to the BBB, whereas most of the nanosystems used in the literature for brain delivery were either of moderate negative charge (−1 to −15 mV) or high negative charge (−15 to −45 mV) which shown the ability to cross the BBB (Saraiva et al., 2016). The aim of this study was to develop new PIP-loaded optimized core-shell NPs to overcome poor drug solubility, enhance its permeability through BBB, and to improve its cytotoxic activity against human brain cancer. The first phase involved PIP nano-micellization using pluronic F127 to optimize solubility, particle size, surface charge and entrapment efficiency. Then, the second phase involved forming PIP-loaded core-shell NPs via coating the PIP-loaded nanomicelles (NMs) with a synthesized positively charged trimethyl chitosan via

Abbreviations: PIP, Piperine; PF127, Pluronic F-127; SDS, Sodium dodecyl sulfate; PVA, Polyvinyl alcohol; CS, Chitosan; TPP, Sodium tripolyphosphate; MTT, Dimethyl sulfate, 3-(4,5dimethylthiazole-2-yl)-2,5-di-phenyl tetrazolium bromide; DMS, Dimethyl sulfate; DCM, Dichloromethane; NMR, Nuclear magnetic resonance spectroscopy; DSC, Differential scanning calorimetry; DLS, Dynamic light scattering; PS, Particle size; PDI, Polydispersity index; ZP, Zeta potential; SEM, Scanning electron microscopy; TEM, Transmission electron microscopy ⁎ Corresponding author at: Nanomedicine Lab, Center for Materials Science, Zewail City of Science and Technology, 6th October City, 12578, Giza, Egypt. E-mail address: [email protected] (I.M. El-Sherbiny). https://doi.org/10.1016/j.ejps.2018.03.030 Received 13 October 2017; Received in revised form 27 February 2018; Accepted 26 March 2018 Available online 27 March 2018 0928-0987/ © 2018 Published by Elsevier B.V.

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Table 1 Evaluation of uncoated and TMC-coated PF17 nanomicelles (NMs). Code

Description

PIP:PF127

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13

Plain NMs PIP-loaded NMs

(0:5) (1:5) (1:10) (1:15)

TMC NPs

TMC-coated plain NMs

TMC-coated PIP-loaded NMs

(0:15) (0:15) (0:15) (1:15) (1:15) (1:15)

TMC:NMs

PS (nm) ± SD

PDI ± SD

Zeta potential (mV) ± SD

(2.5:0) (5:0) (7.5:0) (2.5:1) (5:1) (7.5:1) (2.5:1) (5:1) (7.5:1)

147.3 209.4 216.8 210.2 122.9 109.9 102.5 184.2 151.7 136.5 269.5 308.6 361.2

0.28 0.22 0.13 0.43 0.30 0.38 0.29 0.30 0.39 0.26 0.28 0.29 0.31

−20.8 ± 1.4 −13.6 ± 1.9 −19.8 ± 0.4 −20.5 ± 0.2 15.4 ± 1.4 15.3 ± 0.8 17.5 ± 0.2 18.0 ± 1.5 17.2 ± 1.4 15.4 ± 1.2 12.6 ± 0.8 14.1 ± 1.1 16.4 ± 1.1

± ± ± ± ± ± ± ± ± ± ± ± ±

14.8 4.6 6.7 13.4 14.6 14.4 2.4 23.9 32.8 8.2 33.1 12.5 15.4

± ± ± ± ± ± ± ± ± ± ± ± ±

0.02 0.03 0.03 0.04 0.01 0.03 0.02 0.01 0.06 0.01 0.02 0.02 0.02

water. After centrifugation at 10,000 rpm for 15 min, absorbance of the supernatant was measured using UV–Vis spectrophotometry (Evolution UV 600, Thermo Scientific) at 570 nm against a blank reference. The concentration of the amino groups (mmol/mg) of CS and TMC was then determined by referring to a calibration curve that was determined using series of known concentrations of ethylene diamine. Degree of quaternization (DQ) was then calculated as follows:

electrostatic interaction. The aim of trimethyl chitosan coating was to maintain a positive charge onto the nanocarrier over a wide pH range, confer mucoadhesive properties, and to enhance the permeability through BBB even at neutral pHs. Finally, the cytotoxic activity of the newly developed PIP-loaded core-shell nanosystems on human brain cancer cell line (Hs683) was evaluated via cell viability determination, estimation of IC50, cell cycle arrest analysis, apoptosis assay, and the enzyme-linked immunosorbent assay for CDK2a.

DQ =

[CS − NH 2] − [TMC − NH 2] × 100 [CS − NH 2]

2. Materials and methods 2.1. Materials

2.3. Development of plain and PIP-loaded PF127 nanomicelles

Piperine (PIP, molecular weight of 285.34 Da and purity 98%) was purchased from Alpha Aesar (Ward Hill, MA, USA). Erlotinib was purchased from Sigma-Aldrich (Germany). Pluronic F-127 (PF127), sodium dodecyl sulfate (SDS), polyvinyl alcohol (PVA, Mw 13,000–20,000 Da), chitosan (CS) of Mw 260,000 Da, sodium tripolyphosphate (TPP), DMEM (Invitrogen/Life Technologies), FBS (Hyclone), insulin (Sigma), penicillin-streptomycin, trypsin, EDTA solution, ethanol, acetone, dimethyl sulfate,3-(4,5,-dimethylthiazole-2yl)-2,5-di-phenyl tetrazolium bromide (MTT), dimethyl sulfate (DMS), dichloromethane (DCM) and acetic acid were purchased from SigmaAldrich in China and Germany. Brain cancer cell line, Hs683 (ATCC® HTB-138™) was obtained from American Type Culture Collection. Sodium hydroxide, sodium chloride, and dialysis membranes were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Water purification was achieved using a Milli-Q system (Millipore).

Plain and PIP-loaded PF127 nanomicelles (NMs) were prepared using nanoprecipitation technique (Ali et al., 2016; Basak and Bandyopadhyay, 2013). Briefly, different ratios of PIP and PF127 were used to achieve the suitable size and homogeneity of the micelles, as presented in Table 1 (F1–F4). PIP and PF127 were dissolved in acetone as the organic phase, which was then gradually added to double its volume of distilled water as the aqueous phase. The obtained suspension was stirred till complete evaporation of solvent to produce PF127 NMs powder. 2.4. Development of core-shell nanoparticles via coating of PF127-based NMs Plain and PIP-loaded core-shell NPs were developed via coating of PF127-based NMs with positively charged TMC (Chen et al., 2012; Sheng et al., 2015). Selected NMs were coated with TMC in such a way to optimize the particles overall size and zeta potential (Table 1: F8–F13). Briefly, NMs suspension was gradually added to TMC solution with stirring for 30 min to ensure the deposition of the TMC layer over the NMs followed by dropwise addition of TPP aqueous solution to the mixture while stirring for 30 min at TPP:TMC ratio of 1:10. The nanoparticles were then separated by centrifugation for 30 min at 20,000 rpm at 4 °C followed by lyophilization to obtain dried coated NMs (core-shell NPs). Similar procedures were repeated but without NMs to obtain crosslinked TMC NPs as control formulations (Table 1: F5–F7).

2.2. Synthesis of trimethyl chitosan Trimethyl chitosan (TMC) was synthesized according to Zarifpour et al. procedure (Zarifpour et al., 2013). Briefly, 1 g of CS was dissolved in a mixture of DMS and distilled water (4:1) with stirring for about 10 min. Then, a solution of NaOH and NaCl (1.5:1) was added to the CS solution to deprotonate the nitrogen atom which in turn interacts with the methyl groups from DMS. The mixture was left on a stirrer for 6–8 h till the reaction was completed. Afterwards, the final mixture was purified using a dialysis bag (MW cut-off 12 kDa, Severa) for 3–5 days to obtain pure TMC. The synthesized TMC was characterized by Fourier transform infrared (FTIR) spectroscopy and proton nuclear magnetic resonance spectroscopy (1H NMR). The 1H NMR spectrum was measured in D2O on a 600 MHz spectrometer (Bruker-Biospin, Rheinstetten, Germany). The quaternization degree (DQ) of the synthesized TMC was determined using the standard ninhydrin assay. Briefly, an appropriate amount of CS or TMC was added to 1 mL of sodium acetate/acetic acid buffer solution and 2 mL of 3% ninhydrin agent. The mixture was then heated at 100 °C for 15 min. After cooling to room temperature, the volume of the samples was adjusted to 10 mL using 50% (v/v) ethanol/

2.5. In-vitro characterization of the developed NMs Particle-size, polydispersity-index and zeta-potential were measured using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 °C. Morphological characterization was performed using SEM (Nona Nano SEM, FEI, USA). Also, HR-TEM (JEM-2100F; JEOL, USA) was used to visualize the shape and size of the particles. The specimen was viewed under the microscope at 10–100 k-fold enlargements at an accelerating voltage of 100 kV. Attenuated total reflectance (ATR) spectroscopy was 104

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2.7. Cytotoxicity evaluation of the developed NMs

used for chemical characterization using NECOLET iS10 spectrometer (Thermoscientific, USA) in the range of 600–4000 cm−1 to investigate the principal peaks on lyophilized samples. DSC analysis was performed using DSC-Q20 (TA instrument, USA) to evaluate thermal properties up to 350 °C at a heating rate of 10 °C/min under nitrogen atmosphere (25 mL/min) (Ali et al., 2016). Entrapment efficiency (EE%) and loading efficiency (LE%) were estimated directly using UV–Vis spectrophotometry (Evolution UV 600, Thermo Scientific) at 342 nm using the following equations (Abdellatif et al., 2017). A blank formulation was used as control to eliminate any interference.

2.7.1. Cell viability study Cell viability assay was tested using the MTT assay as reported previously (Senthilraja and Kathiresan, 2015). MTT assay was performed for the free PIP, plain NMs, PIP-loaded NMs, plain TMC-coated NMs and TMC-coated PIP-loaded NMs in addition to erlotinib hydrochloride using a human brain cancer cell line (Hs683) with cells density (1 × 106 cells) and incubation for 24 h at 37 °C. Optical density of the viable cells was assessed using a spectrophotometer by detecting the absorbance of the cell suspension at 595 nm using DMSO as a blank. The cell viability percentage was expressed by the following equation:

EE% = (Entrapped PIP / Total PIP ) × 100

Cell Viability% = (Mean Optical Density / Control Optical Density ) × 100

LE% = (Entrapped PIP / Total NMs ) × 100

2.7.2. Cell cycle study BD FACSCalibur™ (Beckton Dickinson) flow cytometer was used to evaluate the effect of PIP, PIP-loaded NMs and TMC-coated PIP-NMs on the cell cycle of Hs683 cells. All cell cycle phases including pre-G1 (apoptosis), G0/G1, S and G2/M phases were determined by evaluating the cellular uptake of propidium Iodide (PI) via fluorescence-activated cell sorting (FACS) using Hs683 at cells a density (1 × 106 cells/mL) (Liu et al., 2013). Samples were applied to the cells for 24 h and the DNA content analysis was performed using BD FACSCalibur™ (Beckton Dickinson) flow cytometer.

2.6. In-vitro release study The in-vitro release profile of PIP from uncoated and TMC-coated PIP-loaded PF127 NMs (core-shell NPs) was studied using the dialysis bag method against PBS (PH = 7.4) with 1%w/w SDS (Salama and Shamma, 2015). Briefly, in a donor compartment, free PIP, PIP-loaded NMs or TMC-coated PIP-loaded NMs were dispersed in PEG 200 (1.5 mL for each 5 mg of drug) and transferred to dialysis bag (MW cut-off 12 kDa, Severa). The enclosed dialysis bag was immersed in 30 mL of the release medium as a receptor compartment at 37 °C under mild agitation (150 rpm). Aliquots were withdrawn at predetermined time intervals and replaced with fresh release media. Plain NMs and plain TMC-coated NMs were also tested as control formulations. The collected media was filtered through 0.45 μm membrane and the concentration of the released drug was measured using UV–Vis spectrophotometry (Evolution-UV 600, Thermo Scientific, USA) at 342 nm according to the following equation:

2.7.3. Annexin V/propidium iodide apoptosis assay The annexin V/propidium iodide assay was performed on human brain cancer cell line (Hs683) at cells a density (1 × 106 cells/mL) as reported previously (AshaRani et al., 2009; Elbaz et al., 2016). Samples were incubated with cells for 24 h with free PIP, PIP-loaded NMs and TMC-coated PIP-loaded NMs. The staining was carried out as per the manufacturer's instruction (annexin-V FITC apoptosis detection kit, Sigma-Aldrich, St. Louis, MO). The data was collected and analyzed by BD FACSCalibur™ (Beckton Dickinson) flow cytometer.

n−1

Cn = Cn means + A/V

∑ Cs means s=1

2.7.4. Enzyme inhibition study of cyclin dependent kinase-2A Enzyme-linked immunosorbent assay kit for cyclin-dependent kinase 2a (CDK2a) (Cloud-clone Corp, Wuhan, Hubei, China) was used according to the manufacturer's instructions. The plate was examined under the microplate reader (ELISA Reader) and the measurement was conducted immediately at 450 nm.

where Cn is the expected nth sample concentration, Cn means is the measured concentration, A is the volume of withdrawn aliquot, V is the volume of the dissolution medium, n-1 is the total volume of all the previously withdrawn samples before the currently measured sample, and Cs is the total concentration of all previously measured samples before the currently measured sample. Different empirical and mathematical kinetic models were used to fit the release data obtained from the different tested nanosystems (Table 2) using the excel add-in software package (DDSolver) (Zhang et al., 2010) and interpreted according to Costa and Sousa Lobo (2001).

2.8. Statistical analysis All results are expressed in mean ± standard deviation. Significant

Table 2 Mathematical models of the regression for in-vitro release profiles of PIP, PIP-loaded NMs, and TMC-coated PIP-loaded NMs. Model Zero-order First-order Higuchi Korsmeyer-Peppas Hixson-Crowell Hopfenberg Baker-Lonsdale Makoid-Banakar Peppas-Sahlin1 Peppas-Sahlin2 Quadratic Weibull Logistic Gompertz Probit Model parameter a

Equation k0at

F= F = 100a[1-Exp(−k1at)] F = kHat^0.5 F = kKPat^n F = 100a[1-(1-kHCat)^3] F = 100a[1-(1-kHBat)^n] 3/2a[1-(1-F/100)^(2/3)]-F/100 = kBLat F = kMBat^naExp(−kat) F = k1at^m + k2at^(2am) F = k1at^0.5 + k2at F = 100a(k1at^2 + k2at) F = 100a{1-Exp[−((t-Ti)^β)/α]} F = 100aExp[α + βalog(t)]/{1 + Exp[α + βalog(t)]} F = 100aExp{−αaExp[−βalog(t)]} F = 100aΦ[α + βalog(t)]

PIP

PIP-NMs

PIP-NMs/TMC

−2.4870 0.7636 −0.6533 0.6015 −1.1433 0.7636 −0.2521 0.6634 0.6889 0.4149 −1.3666 0.9542a 0.8208 0.8570 0.8070 α = 1.1, β = 0.16, Ti = 2.03

−2.2738 0.9100 0.2384 0.8875 0.5229 0.9099 0.8676 0.9634 0.9605 0.9568 0.1723 0.9710a 0.9694 0.9707 0.9661 α = 1.4, β = 0.52, Ti = 0.915

−4.8968 0.0931 −2.5354 0.6270 −3.1787 0.0930 −1.6444 0.6718 0.7062 −0.5446 −3.4404 0.9736a 0.7312 0.7703 0.7240 α = 1.2, β = 0.15, Ti = 1.99

Symbol refers to best fitting model. 105

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Fig. 1. (a) 1NMR spectrum of TMC, and (b) FTIR spectrum for PIP, plain NMs, PIP-loaded NMs and TMC-coated PIP-loaded NMs.

the formulation with the highest polymer ratio (1:15), and consequently of the higher drug entrapment efficiency, was selected for further investigation. The developed crosslinked TMC-based NPs were fabricated using three different polymer concentrations (F5–F7) which exhibited PS ranged from 102.5 nm to 122.85 nm with PDI around 0.3 and a positive surface charge ranged from 15.3 mV to 17.5 mV (Table 1). On the other hand, coating plain NMs with TMC (F8–F10) showed a significant increase in PS that ranged from 136.53 nm to 184.16 nm. Furthermore, the zeta potential values were switched from negative charges for uncoated NMs (−13.6 to −20.5 mV) to positive charges for TMC-coated NMs (15.4 mV to 18.0 mV). Similar pattern was observed upon coating the PIP-loaded NMs with TMC (F11–F13), where the PS attained was around 2-fold the plain TMC-coated NMs and around 2.8-fold the TMC NPs. On the other hand, the switch of zeta potential to positive values was observed but with less extent (12.6 mV to 16.4 mV). Therefore, F13 was selected for further characterization due to its acceptable size and zeta potential. Kumar et al., 2013 prepared nanoparticles with 443 nm size for brain delivery. (Kumar et al., 2013) FTIR was used to confirm the successful assembly of PIP-loaded NMs as well as the successful formation of TMC-coated PIP-loaded NMs (PIP-loaded core-shell NPs). The IR spectra of plain drug (PIP), plain NMs, PIP-loaded NMs as well as the TMC-coated PIP-loaded NMs were scanned in the range of 1000–4000 cm−1 as shown in Fig. 1b. As apparent from the figure, the PIP spectrum showed characteristic peaks at 3000 cm−1 (aromatic eCeH stretching), 2936.7 and 2852 cm−1 (aliphatic CeH symmetric and asymmetric stretching), 1632.5 cm−1 (stretching of C (carbonyl) eN), 1610.5–1581.4 cm−1 (symmetric and asymmetric stretching of C]C diene), 1502.9 cm−1 (aromatic stretching of C]C), 1436.5 cm−1 (CH2 bending), 1192.9–1250 cm−1 (asymmetric stretching of ]CeOeC), 1030 cm−1 (symmetric stretching of ]CeOeC), 927.5 cm−1 (C(methylene) eO stretching), and at 830–803 cm−1 that corresponds CeH bending. The IR spectrum of PF127 within the developed plain NMs showed an absorption peak at 2880.1 cm−1 that is attributed to the CeH stretching vibration, and a peak at 1342 cm−1 that corresponds to OeH bending as well as another peak noted at 1111 cm−1 that represents the CeOeC stretching vibration. With the aid of the FTIR, the successful assembly of PIP-loaded NMs was confirmed through detecting the characteristic peaks of both PIP and the PF127 in the NMs spectrum. Besides, the successful formation of TMC-coated PIP-loaded NMs (core-shell NPs) was confirmed by the presence of distinctive peaks of PIP, PF127 and TMC together as illustrated in the corresponding spectrum. Successful fabrication of PIP-loaded NMs and TMC-coated PIP-

difference tests were applied like student's t-test and one-way analysis of variance (ANOVA) to all data obtained. All tests were estimated using the software GraphPad Prism Software Version 6. 3. Results 3.1. Synthesis of TMC TMC was successfully synthesized using a single step reaction according to a modified procedure to that adapted by Zarifpour et al. (2013) with a quaternization degree of 96.6% as determined by the ninhydrin assay. The proton nuclear magnetic resonance (1H NMR) spectrum of TMC is shown in Fig. 1a. The signal at 3.22 ppm corresponds to the methyl group at the N, N, N-trimethylated site, while the signal noted at 2.72 ppm is assigned to the methyl group at the N, Ndimethylated site. Finally, the signals ranging from 4.8 to 5.4 ppm are attributed to the hydrogen atom bonded to the C-1 of the glycoside ring which is consistent with the previous report (de Britto et al., 2011). FTIR spectra (Fig. 1b) of the prepared TMC demonstrated the stretching vibration of both NH and OH combined peaks at 3420 cm−1. Besides, the stretching vibration of C]O bond of the acetamido groups was noted at 1658 cm−1, and that of NeH bending of the amino groups and CeH bending of methyl groups were noted at 1564 cm−1 and 1479 cm−1, respectively. 3.2. Characterization of the developed plain/PIP-loaded NMs and core-shell NPs Table 1 demonstrates the particle size (PS) of plain and PIP-loaded NMs at different PIP:PF127 ratios (1:5, 1:10, and 1:15). The mean PS of plain NMs (F1) was 147.26 ± 14.81 nm, while the PIP-loaded NMs (F2–F4) attained PS of 209.37 ± 4.56, 216.80 ± 6.65 and 210.23 ± 13.41 nm, respectively according to their polymer ratio and complemented with PDI values ranged from 0.13 ± 0.03 to 0.43 ± 0.04. On the other hand, the mean ZP of plain NMs (F1) was −20.8 ± 1.4 mV. Furthermore, increasing polymer ratio revealed a significant increase (p-value < 0.01) in surface negativity when comparing F2 with F3 and F4 (−13.6, −19.8 and − 20.5 mV, respectively). The encapsulation efficiency of PIP was found to increase significantly upon increasing PF127 ratio achieving 18.9%, 31.36% and 66.6% for (1:5 w/w), (1:10 w/w) and (1:15 w/w) of PIP:PF127, respectively. The loading efficiency of PIP was found to increase significantly upon increasing PF127 ratio achieving 3.04%, 3.65% and 4.25% for (1:5 w/w), (1:10 w/w) and (1:15 w/w) of PIP:PF127, respectively. Therefore, F4, 106

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Fig. 2. (a) SEM image of TMC-coated PIP-loaded NMs, (b) TEM image of PIP-loaded NMs, and (c) TEM image of TMC-coated PIP-loaded NMs.

nanomicelles exhibited faster dissolution profile than free PIP, where 94.16% was released after 24 h followed by a complete dissolution within 32 h. Coating of PIP-loaded NMs with TMC in form of a coreshell NPs significantly decreased the PIP release with 73.67% of drug released after 24 h followed by a sustained release pattern over 60 days. The release profiles of free PIP and the developed PIP-loaded NMs and TMC-coated PIP-loaded NMs formulations were fitted to different kinetic empirical and mathematical models. All empirical models didn't represent a goodness of fit; therefore, different mathematical models were applied. The best fitting model was Weibull model for the three release profiles with highest r2 (Table 2). This could be attributed to the two phases release profiles with initial fast release followed by slow release pattern (Costa and Sousa Lobo, 2001).

loaded NMs (PIP-loaded core-shell NPs) was also confirmed using both SEM and TEM, as demonstrated in Fig. 2. For instance, Fig. 2a shows the SEM images of TMC-coated PIP-loaded NMs. As apparent from the figure, the TMC-coated PIP-loaded NMs are spherical in shape with almost homogeneous PS distribution which found to be in a good agreement with the results obtained from DLS measurements. Furthermore, TEM images (Fig. 2b) showed spherical PIP-loaded NMs with PS around 100 nm. On the other hand, the TEM micrograph (Fig. 2c) of the TMC-coated PIP-loaded NMs (core-shell NPs) showed a dark core which represents PIP-loaded NMs and a light shell that is attributed to the TMC layer. DSC was also carried out to confirm the successful fabrication of PIP-loaded NMs and TMC-coated PIP-loaded NMs (PIP-loaded coreshell NPs) as well as to particularly detect the success of encapsulation of PIP within the polymer matrices as shown in Fig. 3a. As apparent from the figure, the thermograms of PF127 and TMC showed sharp endothermic peaks at 52.4 °C and 125 °C, representing the PF127 melting and the TMC dehydration, respectively. The TMC thermogram also demonstrated an exothermic peak at about 315 °C that is attributed to the thermal degradation of TMC chains. The DSC thermogram of PIP reveals the appearance of a distinctive endothermic peak at about 130.5 °C that corresponds to its melting point (Yusuf et al., 2013). This peak has disappeared in both PIP-loaded NMs and TMC-coated PIPloaded NMs (core-shell PIP-loaded NPs). Besides, the thermogram of the TMC-coated PIP-loaded NMS (core-shell PIP-loaded NPs) showed a shift in TMC peak from 125 °C to 89 °C, and disappearance of the characteristic peaks of PIP and PF127 which proves the homogenous incorporation of PIP and PIP-loaded NMs in the TMC matrix.

3.4. Cell viability study Cytotoxicity effect of PIP, plain NMs, PIP-loaded NMs, plain NMs/ TMC, TMC-coated PIP-loaded NMs, and Erlotinib, as reference drug, was investigated using MTT assay in brain cancer cell line (Hs683). The cells were subjected to a series of concentrations (0.01 to 100 μg/mL) of the previously-mentioned samples, and then the cell viability was measured after 24 h. The in-vitro results showed a significant decrease in the viability of the cells Hs683 (Fig. 4a) in a dose-dependent manner. The IC50 value of PIP was determined to be 7.06 μg/mL (Fig. 4b). On the other hand, the encapsulation of PIP in nanomicelles (PIP-loaded NMs) decreased the IC50 value significantly (p < 0.05) to 0.67 μg/mL, while the empty nanomicelles attained value was 37.1 μg/mL. Furthermore, coating of PIP-loaded NMs with TMC has significantly (p < 0.05) decreased the IC50 value to 4.02 μg/mL as compared to free PIP but significantly (p < 0.05) increased the value as compared to the PIP-loaded NMs while the plain TMC-coated NMs showed a significant decreasing (p < 0.05) in IC50 value (7.25 μg/mL) than the corresponding plain NMs. IC50 value of Erlotinib was found to be 0.68 μg/ mL which was comparable to PIP-loaded NMs and lower than the coated one (TMC-coated PIP-loaded NMs).

3.3. In-vitro release study and mathematical modeling The in-vitro release study was performed for free PIP, PIP-loaded NMs and TMC-coated PIP-loaded NMs (Fig. 3b,c). According to the figure, free PIP showed a very rapid dissolution as over the first 24 h about 85.2% was dissolved followed by a complete dissolution achieved after 15 days. On the other hand, encapsulation of PIP into PF127-based 107

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in G0-G1 phase which was the highest percentage of cells, 25.69% in S phase, and 6.52% in the G2-M phase. On the other hand, when free PIP was incubated with Hs683 cells, a significant increase (p < 0.05) in pre-G1 phase and G2-M phase with the values of 8.05% and 9.57% attained, respectively. Furthermore, PIP-loaded NMs with and without TMC coating showed a similar pattern like the free PIP but with higher extent. PIP-loaded NMs demonstrated a significant increase (p < 0.05) in both pre-G1 and G2-M phases with the value of 17.09% and 21.41%, respectively. In the case of TMC-coated PIP-loaded NMs, they also showed a significant increase (p < 0.05) in pre-G1 and G2-M phases with values of 13.59% and 18.22%, respectively. 3.6. Annexin V/propidium iodide apoptosis assay This assay was performed to understand the mechanisms by which free and loaded PIP decreased cell proliferation by inducing cell cycle arrest and/or increasing apoptosis. Cell cycle analysis revealed the impact of free and loaded PIP on both pre-G1 (apoptosis) and G2-M phases. Therefore, annexin-V/propidium iodide staining was performed to differentiate apoptosis from necrotic cell death induced by PIP, PIPloaded NMs, and TMC-coated PIP-loaded NMs (Fig. 5f–j). According to Fig. 5j, Hs683 cells didn't show any apoptotic or necrotic events. On the other hand, free PIP has significantly increased (p < 0.05) both apoptotic and necrotic rates where late apoptosis and necrosis were the domain rates. To the same extent, PIP-loaded NMs and TMC-coated PIPloaded NMs demonstrated similar pattern. There was no significant difference between early apoptosis and late apoptosis when comparing PIP-loaded NMs and the TMC-coated PIP-loaded NMs. On the other hand, a significant difference (p < 0.05) between PIP-loaded NMs and TMC-coated PIP-loaded NMs in necrosis was observed. 3.7. Enzyme inhibition To investigate the mechanism that PIP, PIP-loaded NMs and the TMC-coated PIP-loaded NMs arrested the cell cycle in G1 phase; the CDK2a was determined by sandwich enzyme immunoassay using ELISA reader (Fig. 4c). Free PIP at 7.06 μg/mL showed an inhibition for CDK2a of 0.71 μM, while its encapsulation in NMs at 0.67 μg/mL demonstrated a significant decrease to 0.2 μM (Fig. 4d). On the other hand, TMC-coated PIP-loaded NMs at 4.05 μg/mL showed CDK2a inhibition to be 1.85 μM. 4. Discussion TMC is a quaternized derivative of CS which could be synthesized using different reductive methylation approaches (Chen et al., 2013; Kumar et al., 2013). The methylation of CS gives the resulting TMC the ability of maintaining positive charge over a wide pH range, gaining mucoadhesive properties, improving solubility and permeation even at neutral pH at which CS is insoluble and ineffective (Cafaggi et al., 2007). Different methods were used to synthesize TMC including, for instance, the reductive methylation of CS with CH3I in a strong base (Chen et al., 2012). In the present study, TMC was successfully synthesized using a single step reaction according to a modified procedure to that adapted by Zarifpour et al. (2013) with a quaternization degree of 96.6% as determined by the ninhydrin assay. FTIR and 1H NMR have confirmed the addition of methyl groups at the N, N, N-trimethylated site (Fig. 1). Piperine (PIP) has limited therapeutic applications as a result of its poor aqueous solubility. Therefore, loading of PIP into pluronic-based NMs was used to overcome its solubility drawback. Pluronics are triblock copolymers with amphiphilic characteristics due to the hydrophilic polyethylene oxide (PEO) blocks and the hydrophobic polypropylene oxide (PPO) blocks. When pluronic assembled into a micelle form, the hydrophobic core from PPO segments incorporates the hydrophobic drug as the PIP, while the hydrophilic corona from PEO

Fig. 3. (a) DSC thermograms of PIP, PF127, TMC, PIP-loaded NMs and TMCcoated PIP-loaded NMs, and (b) In-vitro release profile of PIP, PIP-loaded NMs and TMC-coated PIP-loaded NMs, and (c) a closer look at the in-vitro release profile within the first 32 h of PIP, PIP-loaded NMs and TMC-coated PIP-loaded NMs.

3.5. Cell cycle study PIP, PIP-loaded NMs and TMC-coated PIP-loaded NMs were evaluated for Hs683 cell cycle by flow cytometry with nuclear propidium iodide staining. The percentage of cells in pre-G1 (apoptosis), G1, S, G2 and M phases was estimated for the previously-mentioned samples with untreated trial as a control (Fig. 5a–d). According to Fig. 5e, Hs683 cells showed 0.58% at the pre-G1 phase (apoptosis), 67.21% of the total cells 108

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Fig. 4. (a) Dose-dependent curve of cell viability percentage of PIP, plain NMs, PIP-loaded NMs, plain NMs/TMC, TMC-coated PIP-loaded NMs, and Erlotinib; (b) IC50 values of PIP, plain NMs, PIP-loaded NMs, plain NMs/TMC, PIP-loaded NMs coated with TMC, and Erlotinib; (c) Dose-dependent curve of cyclin dependent kinase-2A of PIP, PIP-loaded NMs, and TMC-coated PIP-loaded NMs, and (d) Cyclin dependent kinase-2A concentration and IC50 value of PIP, PIP-loaded NMs, and TMC-coated PIP-loaded NMs.

from +12.6 to +16.4 mV for PIP-loaded core-shell NPs, respectively. The attained high positive charge revealed the NMs/NPs stability and prevent their aggregation (Fonseca et al., 2002). The negativity of PIP slightly decreased the overall charge of the TMC-coated NMs. The physical adsorption (through electrostatic interaction) of a TMC layer onto NMs surface was confirmed via comparing the PS of NMs before and after TMC coating. TMC-coated NMs exhibited a significant increase in PS with around 1.5-fold the uncoated NMs which indicated that TMC polymer has successfully coated the NMs forming a shell layer around the NMs spherical core. The resulting core-shell structure was also confirmed visually with the aid of TEM (Fig. 2). Dynamic dialysis technique was adopted for the separation of released PIP from uncoated and TMC-coated PIP-loaded NMs (Fig. 3). The dissolution of the drug mainly directed by solubilization rate and diffusion through dissolution membrane. Piperine is a hydrophobic drug, and although, the sink condition maintained during release study, the solubilization rate allowed 55% of piperine dissolved at 4 h followed by 85% dissolved at 22 h. The remaining 15% mainly affected by particle size which mostly retained the drug in the donor compartment. The dissolution of PIP and release of PIP from uncoated and TMC-coated NMs exhibited a double-phase kinetics model with a fast initial burst release within the first 8 h, followed by a sustained release profile which was expressed by Weibull model. Generally, the parameters that affect the release behavior of a drug from a nanosystem are polymer nature, polymer degradation, drug entrapment efficiency, characteristics of the encapsulated drug and PS and shape (Mittal et al., 2007). Micellization of PIP into pluronic-based NMs enhanced PIP solubility

segments maintains system stability and prevents the micelles aggregation (Zhang et al., 2011). In the current study, nanoprecipitation technique was used to fabricate plain and PIP-loaded pluronic (PF127)based NMs due its simplicity and reproducibility (Basak and Bandyopadhyay, 2013). Different concentrations of PF127 were used in order to optimize the size, charge and drug entrapment. PIP-loaded NMs were successfully fabricated where increasing the polymer, PF127 ratio has improved the overall zeta potential and entrapment efficiency. The mean ZP of plain NMs (F1) was −20.8 ± 1.4 mV, while, incorporation of PIP decreased the charge at low level of pluronic followed by gaining its charge with increasing pluronic ratio. This could be attributed to increasing the PPO hydrophobic core of the NMs which enabled the incorporation of a higher amount of drug, PIP. Furthermore, increasing polymer ratio would improve the nanosystem stability through increasing the system net charge over ± 15 mV. As described in the experimental section, TMC-coated NMs were prepared via dropwise addition of NMs suspension into TMC aqueous solutions with consistent stirring. The TMC concentration demonstrated a significant effect on particles stability which could be returned to the mechanism of polymer bridging (Chen et al., 2012; Mady et al., 2009). The developed plain and PIP-loaded NMs were negatively charged particles ranged from −13.6 to −20.8 mV, while the TMC NPs have been positively charged particles of surface charge ranged from +15.3 to +17.5 mV. When the NMs suspension was added gradually to the TMC solution, the NMs were coated by positive TMC via electrostatic interaction which produced core-shell NPs of net positive surface charge ranged from +15.4 to +18.0 mV for plain core-shell NPs, and 109

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Fig. 5. Hs683 cell cycle evaluated by flow cytometry with nuclear propidium iodide staining. The percentage of cells in pre-G1 (apoptosis), G1, S, G2 and M phases was estimated for PIP (a), PIP-loaded NMs (b), TMC-coated PIP-loaded NMs (c), control cell (d), and comparison between phases of different samples (e), and quadrant dot blot analysis of annexin V/propidium iodide apoptosis assay for PIP (f), PIP-loaded NMs (g), TMC-coated PIP-loaded NMs (h), control cell (i), and comparison between events of different samples (j).

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mechanism that free and loaded-PIP arrested the cell cycle at G1 phase, the cell cycle related protein was determined by sandwich enzyme immunoassay. The experimental results indicated that PIP-loaded NMs induced G1 phase arrest by significantly reducing the expression of CDK2a in Hs683 cell line. The TMC-coated PIP-loaded NMs showed the same effect but to lower extent due to the sustained release profile. The coating of NMs with TMC will support crossing the BBB and improves cancer cell uptake. As a cationic ligand, the TMC facilitates the active transport of nanoparticles via absorption-mediated transcytosis through BBB and because of this; TMC-coated nanoparticles could be used as a drug carrier for brain delivery (Cafaggi et al., 2007; Kumar et al., 2013). Furthermore, the improved anti-tumor efficacy of encapsulated PIP in the optimized formulation makes it a potential candidate for future in-vivo animal study to prove passive targeting through the BBB to brain tumor. Furthermore, clinical translation using intranasal drug delivery system may be investigated.

due to the micellar solubilization of pluronic as amphiphilic polymer. On the other hand, coating the NMs with TMC shell layer has extended the sustained release phase over 60 days. The PIP release mechanism could be expressed as diffusion followed by degradation process, where adding the TMC shell layer was resisting the diffusion of drug from the core-shell nanosystem. The selected uncoated and TMC-coated NMs were further evaluated against human brain cancer cell line (Hs683) to confirm their therapeutic activity. The IC50 values of PIP, PIP-loaded NMs and TMCcoated PIP-loaded NMs were 7.06, 0.67 and 4.02 μg/mL, respectively (Fig. 4). The encapsulation of PIP in NMs enhanced the cytotoxicity of PIP. This could be attributed to the solubilization effect of pluronic which improved the PIP release profile where the available amount of PIP after 24 h was equivalent to 0.268 ng/mL where the DL% is 0.04%, D:P is 1:17.5, EE% is 66.6%, and the percentage release after 24 h was 100%. On the other hand, TMC coating of the NMs has enhanced the cytotoxicity of PIP but to a lesser extent due to the lower available amount of PIP after 24 h (equivalent to 0.15 ng/mL) where the DL% is 0.005%, D:P was 7.5:1, the EE% was 66.6%, and the percentage release after 24 h was 75.23% which was less than the uncoated NMs. This finding was in agreement with a previously published study in which the effect of PIP on human prostate cancer cells was reported (Ouyang et al., 2013). It is worth to mention that TMC as a polymer showed a promising cytotoxic activity where the plain NMs IC50 value was 37.1 μg/mL and that of TMC-coated plain NMs was 7.25 μg/mL which is in agreement with our previously reported study (El-Far et al., 2011) Finally, to understand the mechanisms by which PIP affects cell cycle progression, cell cycle analysis was carried out by staining the DNA with propidium iodide (PI) followed by flow cytometric measurement of the fluorescence (Fig. 5). Cells with damaged DNA will accumulate in one of the phases G1 (Gap 1; accumulates the energy necessary for duplication), S (DNA synthesis; replicates cellular DNA), and G2 (Gap 2; prepares to divide), and the mitotic phase (M; cell division) with three checkpoints which are G0/G1, S and G2/M phases. Cells with irreversible damage will undergo apoptosis, giving rise to accumulation of cells in pre-G1 phase (AshaRani et al., 2009; Cobb, 2013; Ocak et al., 2012). Thus toxicity studies were further extended to cell cycle analysis to detect parameters such as apoptosis, cell cycle arrest and enzyme inhibition. Free PIP and NMs showed G2 arrest which was observed as an increase in cell population in G2/M phase compared to control. In control, major cell population was observed in G1 and S phases while both uncoated and coated NMs showed a decrease in G1 and S population accompanied by an increase in G2/M population. Furthermore, a significant apoptosis was observed, as indicated by the increase of cell population in pre-G1. Similar results were reported for another natural product (curcumin) (Liu et al., 2007). To assess the extent and mode of cell death, annexin V/propidium iodide staining was carried out. From the dot plots chart data were statistically extracted based on the percentages of unstained cells (viable cells), red fluorescent labeled cells (necrotic cells), green labeled cells (apoptotic cells), and dual stained cells (late apoptosis). The data from the annexin V/propidium iodide staining experiment indicated that a small percentage of cells were undergoing apoptosis and necrosis. However, a tendency for late apoptosis and necrosis was observed especially for the PIP-loaded NMs and TMC-coated PIP-loaded NMs. But the significant increases in the percentage of apoptosis in cell cycle analysis and both late apoptosis and necrosis in annexin V/propidium iodide staining experiment suggested that PIP played its inhibitory roles on brain cancer cells mainly via the induction of cell cycle arrest and apoptosis. The cyclins and cyclin-dependent kinases (CDKs) are positively regulating cell cycle progression (Węsierska-Gądek and Kramer, 2011). In both S phase and G2/M transition in the cell cycle, cyclin A plays a crucial role while in G1 to S phase, cyclin D1 regulates the progression. The cyclin-CDK complexes are involved in different periods of the cell cycle. The formation of cyclin A and CDK2 dimer complex initiates S phase (Schwartz and Shah, 2005). Therefore, to investigate the

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