Stable phosphatidylcholine-bile salt mixed micelles enhance oral absorption of paclitaxel: preparation and mechanism in rats.

http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(10): 901–912 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2014.945090

ORIGINAL ARTICLE

Stable phosphatidylcholine-bile salt mixed micelles enhance oral absorption of paclitaxel: preparation and mechanism in rats Yanli Zhao*, Yanan Cui*, Yimu Li, and Lingbing Li

Journal of Drug Targeting Downloaded from informahealthcare.com by McMaster University on 12/26/14 For personal use only.

Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province, China

Abstract

Keywords

The aim of this study is to prepare a stable phosphatidylcholine/bile salt micelles with Pluronic F127-polyethylenimine conjugates (F127-PEI), D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS), soybean phosphatidylcholine (SPC) and sodium cholate (NaC) and to elucidate the effects and possible mechanism of micelle components on the intestinal absorption of paclitaxel (PTX) in rats. The results of intestinal absorption revealed that the PTX in SPC/NaC micelles displayed superior permeability across intestinal barrier than free drug and PTX in TPGS/SPC/NaC and F127-PEI/TPGS/SPC/NaC mixed micelles exhibited the strongest permeability across intestinal barrier. These results were also proved by the studies on cell uptake tests. The mechanism was demonstrated in connection with inhibition of the efflux mediated by intestinal P-gp and enhancement of the drug transportation across the unstirred water layer to the endothelial lining, thereby promoting the permeation across the intestinal wall. Pharmacokinetic study demonstrated that the area under the plasma concentration–time curve (AUC0!1) of paclitaxel in F127-PEI/TPGS/SPC/NaC micelles was much greater than that in TPGS/SPC/NaC micelles. This phenomenon deviated from the results of uptake studies by cells and permeability experiments through rat intestine and revealed that the micelle stability had a great effect on intestinal absorption of paclitaxel.

Mechanism, oral administration, paclitaxel, phosphatidylcholine-bile salt mixed micelles, pluronic-polyethylenimine, TPGS

Introduction Paclitaxel (PTX) is an effective anti-tumor drug extracted from Taxus chinensis and widely used in treating a broad range of tumors such as ovarian cancer, breast cancer and colorectal cancer. However, in clinical treatment, the traditional application of PTX is extremely limited to parenteral administration. Oral bioavailability of PTX is even less than 10% [1] as the results of the poor solubility (about 300 ng/ mL), the action of the multidrug efflux transporter P-glycoprotein (P-gp) highly expressed in intestinal tract as well as the extensive first-pass metabolism by either the intestinal or liver cytochrome P450 enzymes like CYP3A4 [2]. In the past years, many strategies have been contributed in exploring an alternative oral delivery system for PTX to not only improve its poor solubility and low permeability across the intestinal barrier but also overcome the multidrug resistance [3–5]. Aguëros et al. promoted the oral bioavailability significantly by encapsulating PTX into

*The first two authors contributed equally to this study. Address for correspondence: Lingbing Li, Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong Province 250012, China. Tel: +86-531-88382015. Fax: +86-531-88382548. E-mail: [email protected]

History Received 22 April 2014 Accepted 12 July 2014 Published online 31 July 2014

poly(anhydride) nanoparticles [6]. Yoncheva et al. established superior PTX Pluronic micelles, achieved a higher AUC0–1 area and longer mean residence time, indicating an efficient oral absorption of PTX [7]. Phosphatidylcholine/bile salts mixed micelle system (PC/BS-MM) is generally accepted to be a good candidate for drug delivery system because of its physiological compatibility and solubilizing capacity [8–10]. In intestine, the PC/BS mixed micelles can deliver hydrophobic drugs across the barrier of unstirred water layer to the enterocytes and increase their permeability across the intestinal membrane [11]. However, due to the large steroidal skeleton of bile salts which hinders the micelle aggregation, the structure of PC/BS micelles is unstable and drug releases from micelles easily [12]. To solve these problems, modified PC/BS mixed micelles attracted much attention [13]. D-a-Tocopheryl polyethylene glycol 1000 succinate (TPGS) comprised of a hydrophilic portion and a lipophilic portion, can self-assemble to micelles to improve solubility of hydrophobic drugs as well as inhibit P-gp activity to increase the intestinal absorption [14]. Varma et al. confirmed TPGS can inhibit the role of P-gp-mediated efflux and enhance the oral absorption of paclitaxel [15]. Polyethylenimine (PEI) was a common basis of polymeric carriers which have been widely used in drug delivery systems, especially gene delivery [16]. Furthermore, cationic PEI polymers can enhance the electrostatic interaction of micelles with negatively charged site on


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tumor cell surface and intestinal epithelial cells, promoting the uptake in resistant tumor cells and intestinal absorption. In our previous studies, Pluronic F127-polyethylenimine copolymers (F127-PEI) have been synthesized by conjugating branched PEI with CDI-activated Pluronic F127 [17]. In this study, F127-PEI was incorporated into the soybean phosphatidylcholine/sodium cholate (SPC/NaC) mixed micelle system. Through the electrostatic interaction between F127-PEI and NaC and steric protection of long PEO segment in F127 molecule, the micelles could aggregate more tightly and structure could become more stable. TPGS, an inhibitor against P-gp, was also incorporated into the formulation to improve the intestinal absorption [15]. Systematic studies on physicochemical properties including size distribution, zetapotential and morphology were conducted to validate the formation of micelle structure. CMC and release behavior were then studied. To clarify the possible absorption mechanism in the intestine, the effects of micelle components on the absorption of PTX were investigated using permeability studies through rat intestinal barrier, cell-uptake test against resistant breast cancer cells and pharmacokinetic study in rats.

Materials and methods Materials Pluronic F127-polyethylenimine conjugates (F127-PEI) were synthesized by conjugating branched PEI with CDI-activated Pluronic F127 according to our previous report [17]. Paclitaxel (PTX) was purchased from Chengdu Furunde Industrial Co. Ltd. (Chengdu, China). Soybean phosphatidylcholine (SPC, injection grade) was supplied by Shanghai Taiwei Medicine Co. Ltd. (China). TPGS, sodium cholate (NaC), pyrene and rhodamine-123 (Rh-123) were purchased from Sigma-Aldrich (St. Louis, MO). Pentobarbital sodium was purchased from Guangzhou Chemical Reagent Co. Ltd. (Guangzhou, China). Verapamil and DMSO were purchased from Siyou Chemical Reagent Co. Ltd. (Tianjin, China). All other reagents and buffer solution components were analytical grade preparation. Distilled and deionized water was used in all experiments. Cell culture Human breast carcinoma MCF-7 cells and doxorubicinresistant breast cancer MCF-7/Adr cells (obtained from Institute of Immunopharmacology, Shandong University, China) were maintained in RPMI-1640 (Macgene Biotech Co., Ltd, Beijing, China) supplemented with 10% heatinactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 U/mL). To maintain the drug resistance phenotype, it was cultured in the presence of 2 mM doxorubicin and passaged for 1 week in a drug-free medium before the experiment. The cells were cultured at 37 C in humidified atmosphere with 5% CO2. Preparation of PTX-loaded mixed micelles PTX-loaded TPGS/SPC/NaC mixed micelles were prepared using film hydration method [2]. Briefly, 2.5 mg of PTX, 7.57 mg (5 mmol) TPGS, 4.3 mg (5 mmol) SPC and 2.6 mg (5 mmol) NaC were dissolved in 3 mL dehydrated ethanol in a

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round bottom flask. The organic solvent was removed by rotary vacuum evaporation and the transparent film was obtained after vacuum dehydration for 24 h. The dry film was hydrated by distilled water for 30 min and the solution was filtered by 0.22 mm nylon filter to remove any precipitated drug. F127-PEI/TPGS/SPC/NaC mixed micelles were prepared under the same procedure described above with 3.2 mg F127-PEI (20% of total carrier materials, w/w) concluded in the distilled water. Size distribution and zeta-potential measurement Size distribution and zeta-potential of the micelles were measured using a dynamic light scattering (DLS) instrument (Zetasizer-3000, Malvern Instruments, Malvern, UK) equipped with a He–Ne laser of 633 nm at a fixed angle of 90 at 25 C. These two measurements were performed in triplicate at a polymer concentration of 6 mmol/L. Transmission electron microscopy for morphology Morphological features of the micelles were observed by transmission electron microscope (TEM) using a JEM-100CX electron microscope (JEOL, Tokyo, Japan). After diluted to a concentration of about 6 mmol/L with distilled water, the samples were negatively stained with 2% (w/v) phosphotungstic acid for observation. Drug-loading content The amount of PTX encapsulated in micelles was measured by RP-HPLC assay according to the reported method with modifications [18]. First, 200 mL micelle solution was diluted with 800 mL acetonitrile to destroy micelle structure and release PTX. Mixed solution was then filtered by 0.22 mm nylon filter. The mobile phase was acetonitrile/water (50:50, v/v) and the detection wavelength was 227 nm. The standard regression equation was A ¼ 46.427 C 8.5321, (r2 ¼ 0.9999). Drug-loading content (DL%) and encapsulation efficiency (EE%) were calculated by the following equations: 0 1 B DL% ¼ B @

C weight of drug in micelles C 100% weight of micelle materials added A þweight of drug added

EE% ¼

weight of drug in micelles 100% weight of drug added

Critical micelle concentration determination Critical micelle concentration (CMC) values were detected with pyrene as a fluorescence probe according to previous procedure with modifications [19]. Briefly, 100 mL pyrene/ acetone stock solution (2.0 10 4 mol/L) was added into a series of glass centrifuge tubes, respectively. After acetone was drying in the nitrogen gas flow, 10 mL drug-unloaded micelle solutions at various concentrations were added into the tubes with a final concentration of pyrene at 2.0 106 mol/L. The mixture was incubated for 5 h at 45 C, then overnight at 25 C. The samples were measured by

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F-2500 fluorescence spectrometer (Hitach, Tokyo, Japan) with the emission wavelength of 350–500 nm and excitation wavelength of 334 nm. Ratio of fluorescence intensity at 373 nm (I1) and 384 nm (I3) was plotted according to the concentration of micelles. CMC values were observed corresponding to the bump of the curve. Storage stability Storage stability of micelles was evaluated by monitoring the time-dependent changes of physical characteristic after storage at 4 C for 48 h.


Stability study in simulated gastrointestinal fluid In order to validate the stability of micelles in digestive fluid, stability study was tested in two different kinds of simulated gastrointestinal fluids: SGF containing pepsin (0.32%, w/v) with a pH of 1.2, SIF containing pancreatin (1%, w/v) with a pH of 6.8 [20]. Briefly, 2 mL PTX-loaded micelle solutions were diluted with 8 mL simulated gastrointestinal fluids before incubating in water bath for 120 rpm at 37 C. Samples were collected at given time of 0, 0.5, 1, 2 h for the test in SGF and 0, 0.5, 1, 2, 3, 6 h for SIF. Size distributions and relative encapsulation efficiencies, compared to initial ones, were studied after filtration by 0.22 mm nylon filter to remove insoluble enzyme and PTX crystallized. In vitro release of mixed micelles In vitro release behavior of PTX from micelles was investigated using dynamic state membrane dialysis method [21]. This part of experiment was conducted in simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8), respectively. To meet the sink condition, Tween-80 was included in the release mediums as a solubilizer (1%, w/v). The samples, containing 80 mg PTX, were added in dialysis bags with the cut-off molecular weight of 3500 and the bags were placed into 20 mL release medium and incubated for 120 rpm at 37 C. At predetermined interval (0.25, 0.5, 075, 1, 2, 4, 6, 8, 10, 12, 24 and 48 h), 2 mL release medium was taken out for further determination and fresh release medium was added in with the same volume and temperature. Cytotoxicity of empty micelles The cytotoxicity of empty micelles, in contrast with that of F127-PEI copolymers, was examined with MCF-7/Adr cells by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT). Briefly, the cells were seeded into 96-well plate at 5000 cells/well and cultured at 37 C for 24 h in the presence of 5% CO2. The cells were then exposed to a series of concentrations of empty micelles and F127-PEI copolymers, respectively. The blank culture medium (absence of cells) was used as a blank control. After incubation for 48 h, 20 mL MTT solution (0.5 mg/mL) was added in the plate and the mixture was incubated for further 4 h. To dissolve the violet crystals formed, the supernatant was removed after centrifugation and 150 mL DMSO was added. Optical density (OD) was recorded at 570 nm by an ELISA microplate reader

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(Bio-Rad, Hercules, CA). The relative cell viability was calculated as follows: Cell viabilityð%Þ ¼

OD570ðsampleÞ 100% OD570ðcontrolÞ

where OD570(control) was obtained in the absence of the copolymers or empty micelles, while OD570(sample) was obtained in the presence of the copolymers or empty micelles, both blank deducted. Uptake by resistant breast cancer cells In this part of study, rhodamine-123, a fluorescent dye and a substrate of P-gp efflux pump, was chosen as the model drug. The intracellular uptake in MCF-7/Adr cells was detected by fluorescence microscopy as well as flow cytometry with different rhodamine-123 formulations [22]. In brief, MCF-7/Adr cells were seeded into 6-well culture plates at 2 105 cells/well and cultured at 37 C for 24 h in the presence of 5% CO2. The cells were then incubated with different rhodamine-123 formulations and the concentrations of rhodamine-123-loaded or solubilized micelles were all the same at 5 mmoL/L. After incubation, the cells were washed thrice with cold PBS to remove the rhodamine-123 formulations unabsorbed. The intracellular uptake of rhodamine-123 was qualitatively observed by fluorescence microscopy (OLYMPUS, Tokyo, Japan). For a quantitative study, after the rhodamine-123 formulations unabsorbed were washed by cold PBS, 500 mL trypsin PBS solution (2.5 mg/mL) was added into the plates. The cells were trypsinized for 1 min and then harvested by adding 2 mL complete culture medium. Intracellular uptake of rhodamine123 in the cells was then detected using flow cytometry (FCM, BIO-RAD). Permeability of PTX in micelles through rat intestine This part of test was carried out based on a reported method [23] and all animal procedures were approved by the Shandong University Animal Care and Use Committee. Healthy male Wistar rats weighted 250 ± 20 g were used and each of the rats had been fasted for 24 h, free access to water, before experiment. To start the test, rats were anesthetized with an intraperitoneal injection of l% sodium pentobarbital (dosage, 0.4 mL/100g) and the abdominal wall was opened. Carefully, the whole small intestine from the beginning of duodenum to the end of ileum was isolated in situ and ligated at both ends with canulas. Warm saline was passed slowly through the tract to clear the gut. After gut cleared, the PTX formulations, diluted in Krebs–Rings solution (K-R, pH 6.8, NaCl 7.8%, CaCl2 0.37%, KCl 0.35%, MgCl 20.02%, NaH2PO4 0.32%, glucose 1.48%, NaHCO3 1.37%), were perfused circularly through the intestine at an entering flow rate of 0.25 mL/min. A heating lamp was used to maintain the body temperature of the rats throughout the experiment. Upon the whole selected intestine fed up with perfusion solution, the time point was recorded as T0. At given times (0.25, 0.5, 0.75, 1.0, 1.25 and 1.5 h), entering perfusion samples were taken from the cylinder while exiting ones collected continuously for 30 s into pre-weighed 5-mL glass vials from tail end

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of the perfusate. Content of PTX in the entering and exiting perfusion was then determined by HPLC analysis and exiting flow rate of the perfusion was calculated through the weight of the perfusion collected and the interval of the time. To calculate the surface area of the intestinal segment studied, rats were killed after experiment and the length and diameter of the selected intestine were measured. The permeability rate constant (Ka) and effective permeability coefficient (Peff) were calculated with the following equations:

of PTX for the intravenous and oral administration, respectively. Statistical analysis Results are given as mean ± SD. Statistical analysis was performed using Student’s t-test. Pharmacokinetic parameters were calculated using a non-compartmental model by the software of Drug and Statistics (DAS, version 2.0). Statistical significance was tested by two-tailed Student’s t-test. Statistical significance was set at p50.05.

Ka ¼ ð1 Cout Qout =Cin Qin ÞQ=V

Results


Peff ¼ ½Qin lnðCout Qout =Cin Qin Þ=A where Qin and Qout are the flow rate (mL/min) of entering and exiting perfusion, respectively; Cin and Cout were the concentration (mg/mL) of PTX in the entering and exiting perfusion at given times, respectively; Q is the measured perfusion rate (0.25 mL/min); V is the volume (cm3) and A is the surface area (cm2) of the intestinal segment studied. At each given time, Ka and Peff are calculated, respectively, and the mean value is lastly figured. Pharmacokinetic studies in rats The in vivo pharmacokinetic study was carried out with healthy male Wistar rats weighted 250 ± 20 g. The rats were fasted overnight with free access to water before different PTX formulations were administrated. At given times (0.0833, 0.25, 0.5, 1, 2, 4, 6, 8 h for injection group and 0.25, 0.5, 1, 2, 4, 6, 8, 12 h for oral groups), 0.3 mL blood was drawn from the subclavian vein, placed into heparinized tubes and separated immediately by centrifugation (4000 rpm for 15 min). After centrifugation, the plasma obtained was stored at 20 C until analysis [24]. To precipitate proteins in the plasma, 150 mL of rat plasma was added into 750 mL precipitant solvent (methanol/acetonitrile, 5/5, v/v), vortexed for 5 min, and then centrifuged for 10 min at 10 000 rpm. The supernatant was transferred into another glass tube and evaporated by nitrogen flow at 40 C. Afterwards, the extraction residual was re-dissolved in 100 mL acetonitrile and vortexed for 3 min before centrifuged for 5 min at 10 000 rpm. The concentration of PTX in plasma was determined by HPLC analysis. Calibration curve for the concentration range of 0.03–2 mg/mL was A ¼ 95.754c 2.5792 (r2 ¼ 0.9997). The average recovery was 101.53 ± 1.975% and the coefficients of variation (CV) for within and between days were 3.82 and 4.46%, respectively. The pharmacokinetic parameters were calculated using the software of Drug and Statistics (DAS, version 2.0). In addition, the absolute bioavailability (F%) of PTX from different oral formulations was examined as the following equation: F% ¼

ðAUCoral Di:v: Þ 100% ðAUCi:v: Doral Þ

where AUCoral and AUCi.v. were the areas under the plasma concentration–time curve of PTX for the oral and intravenous administration, respectively; Di.v. and Doral were the dosages

Development and characterization of mixed micelles To improve the stability and the oral bioavailability of SPC/ NaC mixed micelle system, the stable micelles made of F127-PEI, TPGS, SPC and NaC were prepared. Through the electrostatic interaction and steric protection of long PEO segment in F127 molecule, the micelle aggregation became more tightly and the stability was improved (Figure 1). The TEM results in Figure 2 showed the micelle spherical in shape and good dispersibility. Furthermore, the TEM results revealed the difference between the structure of TPGS/ SPC/NaC micelles and F127-PEI/TPGS/SPC/NaC micelles. The structure of SPC/NaC micelles is widely accepted as a ‘‘mixed disk model’’ [25]. In this model, the mixed micelle consists of a disk-like portion of a phospholipid bilayer surrounded at its perimeter by bile salt molecules. TEM picture of TPGS/SPC/NaC micelles in Figure 2(A) showed a similar structure with that of SPC/NaC micelles. While the F127-PEI/TPGS/SPC/NaC micelles (Figure 2B) showed a distinctly shell, indicating the F127-PEI was incorporated into micelles and the long hydrophilic shell made of PEO segment in F127 molecule was formed. Particle diameter and zeta-potential of the micelles were measured using DLS. Results showed F127-PEI/TPGS/SPC/ NaC micelles a smaller diameter of 48.8 ± 0.62 nm contrasted with TPGS/SPC/NaC micelles (54.4 ± 1.58 nm). As for zetapotential, TPGS/SPC/NaC micelles displayed negative values of 39.5 ± 0.82 mV while F127-PEI/TPGS/SPC/NaC micelles showed increased ones to 1.5 ± 0.46 mV due to the participation of amine group in F127-PEI (Table 1). Drug encapsulation efficiency and loading content For TPGS/SPC/NaC micelles, when the molar ratio was 1:1:1, the drug-loading content was the highest (data not shown). Thus, this molar ratio (1:1:1) was used as the optimal molar ratio of material components in preparation section. For F127-PEI/TPGS/SPC/NaC micelles, when the weight fraction of F127-PEI was 20%, the zeta-potential of micelles were approximately neutral and the drug loading content was the highest (data not shown). Thus, this fraction (20%) was used as the optimal fraction in preparation section. Drug encapsulation efficiency and loading content of the micelles under optimal formulation were then measured in triplicate in independent experiments with HPLC analysis. Drug-loading content (DL%) and encapsulation efficiency (EE%) of F127-PEI/TPGS/SPC/NaC micelles were 10.91 ± 0.29 and 95.2 ± 2.4%, respectively. As for TPGS/SPC/NaC micelles,


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Figure 1. The schematic representation of the formation of F127-PEI/TPGS/SPC/NaC micelles.

Figure 2. TEM micrographs of (A) TPGS/SPC/NaC micelles and (B) F127-PEI/TPGS/SPC/NaC micelles.

NaC and steric protection of the PEO segment in F127 molecule.

Table 1. Characteristics of the micelles (n ¼ 3).

Formulation Diameter (nm) DL (%) EE (%) CMC (mol/L) Zeta potential (mV)

F127-PEI/TPGS/ SPC/NaC micelles

TPGS/SPC/ NaC micelles

48.8 ± 0.62 10.91 ± 0.29 95.2 ± 2.4 1.2 106 1.5 ± 0.46

54.4 ± 1.58 8.53 ± 0.35 71.2 ± 2.6 3.5 105 39.5 ± 0.82

the DL% and EE% were respectively 8.53 ± 0.35 and 71.2 ± 2.6%, respectively. When F127-PEI copolymers was introduced, the micelle system showed better ability of drug loading capacity (Table 1). CMC value of mixed micelles CMC values of two types of micelle systems were evaluated with pyrene as a probe. The result showed a CMC value of about 1.2 106 mol/L for F127-PEI/TPGS/SPC/NaC micelles and about 3.5 105 mol/L for TPGS/SPC/NaC micelles (Table 1). The CMC value of F127-PEI/TPGS/ SPC/NaC micelles was lower than that of TPGS/SPC/NaC micelles due to the electrostatic interaction between PEI and

Storage stability The drug-loaded F127-PEI/TPGS/SPC/NaC mixed micelles were stable during storage at 4 C for 48 h. No precipitation of drug or micelle size/size distribution changes was observed during this period. However, for TPGS/SPC/NaC mixed micelles, the precipitation occurred after 4 h (data not shown). This is due to the large steroidal skeleton of NaC which hindered the aggregation of micelles and caused the drug release from micelles [12]. While in the case of F127-PEI/TPGS/SPC/NaC mixed micelles, the electrostatic interaction between PEI and NaC and steric protection of the PEO segment made the micelles more stable. Stability of mixed micelles in simulated gastrointestinal environment A severe difficulty encountered with PTX-loaded micelles after oral administration was the instability in complex gastrointestinal environment. The stability of PTX-loaded F127-PEI/TPGS/SPC/NaC micelles in gastric juice was

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evaluated in SGF concluding 0.32% (w/v) pepsin, simulating fed conditions in the stomach. The results showed PTXloaded F127-PEI//TPGS/SPC/NaC and TPGS/SPC/NaC micelles were stable in SGF. The size did not change obviously within 2 h (Figure 3A and B) and relatively 12.2 and 19.3% of PTX-loaded micelles were released, respectively (Figure 4A and B). The stability in SIF was also studied. In the presence of pancreatin, the size of TPGS/SPC/NaC micelles was changed obviously (Figure 3D). Meanwhile, a release of 83% was observed in Figure 4D. However, in the case of F127-PEI/ TPGS/SPC/NaC micelles, the size did not change obviously within 6 h (Figure 3C) and relatively 30% of PTX-loaded micelles were released (Figure 4C). Release behavior of PTX from mixed micelles In vitro release of PTX from micelles was evaluated by dialysis method using a dialysis bag with a molecular weight cut-off of 3500 Da. To meet the sink condition, SGF and SIF containing 1% (w/v) Tween-80 were chosen as the release medium. Figure 5 demonstrated two kinds of PTX-loaded micelles, compared with PTX solution (dissolved in 1:1 Cremophor EL/ethanol), both showed controlled-release properties. For PTX solution, above 95% of drug was released in both release mediums within 12 h. While for PTX micelles, only 20% was released within 2 h and less than 80% within 12 h. Results also showed that in both mediums, PTX-loaded F127-PEI/TPGS/SPC/NaC micelles exhibited faster release characteristics than TPGS/SPC/NaC ones. This result might be attributed to the fact that with thicker hydrophilic shell, PTX-loaded F127-PEI/TPGS/SPC/NaC micelles facilitated more channels for diffusion of water into the core and subsequent diffusion of PTX out of the micelles.

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Cytotoxicity of empty micelles The cytotoxicity of empty F127-PEI/TPGS/SPC/NaC micelles was evaluated by cell viability assay with MCF-7/ Adr cells, in comparison with F127-PEI copolymers. As shown in Figure 6, with the increase concentration of F127-PEI copolymer and empty micelles, viability of MCF-7/Adr cells decreased accordingly. However, the empty F127-PEI/TPGS/SPC/NaC micelles showed an obviously lower cytotoxicity than F127-PEI copolymers and above 90% of the MCF-7/Adr cells were still alive after 48 h of incubation at the concentration of 2 mg/mL. Uptake by resistant breast cancer cells Figures 7 and 8 show the fluorescence images and flow cytometry analysis of MCF-7/Adr cells after incubating for 2 h with rhodamine-123 solution (DMSO51%, v/v), rhodamine-123 solution with verapamil (DMSO51%, v/v), rhodamine-123-loaded SPC/NaC micelles, rhodamine-123-loaded SPC/NaC micelles with verapamil, rhodamine-123-loaded TPGS/SPC/NaC micelles, rhodamine-123-loaded TPGS/SPC/ NaC micelles with verapamil, rhodamine-123-loaded F127-PEI/TPGS/SPC/NaC micelles and rhodamine-123loaded F127-PEI/TPGS/SPC/NaC micelles with verapamil, respectively. Contrasted with rhodamine-123 dissolved in DMSO solution, the formulation with verapamil displayed more fluorescence intensity due to the P-gp efflux pump inhibition effect of verapamil. Similar phenomenon emerged between rhodamine-123 SPC/NaC micelles with and without verapamil. However, fluorescence intensity showed barely enhancement between rhodamine-123-loaded TPGS/SPC/ NaC micelles with and without verapamil as well as the rhodamine-123-loaded F127-PEI/TPGS/SPC/NaC ones with and without verapamil, indicating TPGS a potential inhibitor against P-gp.

Figure 3. Changes of the size of PTX-loaded F127-PEI/TPGS/SPC/NaC and TPGS/SPC/NaC micelles during incubation in SGF and SIF: (A) F127PEI/TPGS/SPC/NaC micelles in SGF; (B) TPGS/SPC/NaC micelles in SGF; (C) F127-PEI/TPGS/SPC/NaC micelles in SIF; (D) TPGS/SPC/NaC micelles in SIF (n ¼ 3).


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Figure 4. Relative percentage of PTX encapsulation efficiency of the F127-PEI/TPGS/SPC/NaC and TPGS/SPC/NaC micelles corresponding to that of the initial ones during incubation in SGF and SIF. (A) F127-PEI/TPGS/SPC/NaC micelles in SGF; (B) TPGS/SPC/NaC micelles in SGF; (C) F127-PEI/ TPGS/SPC/NaC micelles in SIF; (D) TPGS/SPC/NaC micelles in SIF (n ¼ 3).

Permeability of PTX in micelles through rat intestine To investigate the penetrating quality of PTX-loaded micelles, permeability study was conducted in rat intestine. As listed in Table 2, the Ka and Peff of PTX in F127-PEI/TPGS/SPC/NaC micellar solutions in the whole rat small intestine under low (25 mg/mL), middle (50 mg/mL) and high (100 mg/mL) concentrations displayed concentration-independent changes, which may indicated intestinal absorption of PTX in F127-PEI/TPGS/SPC/NaC mixed micelles as passive transfer by diffusion across lipid membranes. In order to further investigate the respective effect of F127-PEI, NaC and TPGS in PTX intestinal permeability, four different formulations, PTX solution dissolved in a 50/50 (v/v) mixture of Cremophore EL/dehydrated ethanol, PTX-loaded SPC/NaC micelles, PTX-loaded TPGS/SPC/ NaC micelles and PTX-loaded F127-PEI/TPGS/SPC/NaC micelles were evaluated through intestinal permeability tests in situ. The concentrations of PTX in each group were all the same at 50 mg/mL and the results were listed in Table 3. As is shown, the Ka and Peff of PTX in SPC/NaC micelles were significantly higher than those of PTX in solution (p50.05). The permeability of PTX in TPGS/SPC/NaC ones was increased significantly (p50.05) compared with PTX in SPC/NaC micelles. However, the permeability of PTX showed barely enhancement between F127-PEI/TPGS/SPC/NaC micelles and TPGS/SPC/NaC micelles.

Permeability study in various intestinal segments was also carried out. The results showed Ka of (2.76 ± 0.82) 103 min1, (3.11 ± 0.98) 103 min1, (2.02 ± 0.72) 103 min1, (1.84 ± 0.73) 103 min1 and Peff of (4.96 ± 1.44) 102 cmmin1, (5.61 ± 1.73) 102 cmmin1, 2 1 (3.61 ± 1.27) 10 cmmin , (3.28 ± 1.29) 102 1 cmmin for duodenum, jejunum, ileum and colon, respectively, indicating a site-independence in the permeability of PTX in F127-PEI/TPGS/SPC/NaC micelles. Pharmacokinetics studies in rats The in vivo pharmacokinetic study in rats was carried out in five groups. One group was injected with PTX solution dissolved in a 50/50 (v/v) mixture of Cremophore EL/ dehydrated ethanol and the other four groups were orally administered with four different types of formulations, respectively: PTX solution dissolved in a 50/50 (v/v) mixture of Cremophore EL/dehydrated ethanol, PTX solution with verapamil, PTX-loaded TPGS/SPC/NaC micelles and PTX-loaded F127-PEI/TPGS/SPC/NaC micelles. The dosage of PTX was 4 mg/kg for injection and 20 mg/kg for oral administration. Plasma concentration–time profiles and pharmacokinetic parameters of PTX in different formulations were presented in Figure 9 and Table 4, respectively. As is shown, the oral absorption of PTX in solution was extremely limited with an absolute bioavailability of 10.56%. Co-administration of PTX

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administered orally with and without verapamil, PTX-loaded TPGS/SPC/NaC micelles resulted in a significant improvement in plasma concentration. The absolute bioavailability of PTX was increased to 28.93%, indicating the potential P-gp inhibition effect of TPGS similar with verapamil and promoting across the unstirred water layer effect of NaC, which was consistent with the results of uptake studies by cells and permeability experiments through rats intestine. More importantly, PTX in F127-PEI/TPGS/SPC/NaC micelles displayed highest AUC0–1 (3387 mgh/L) and Cmax (282 mg/L) and the absolute bioavailability was also increased to 41.15 ± 4.19%.


Discussion Development and characterization of drug-loaded micelles

Figure 5. Cumulative release percentage of PTX from PTX solution (^), PTX-loaded F127-PEI/TPGS/SPC/NaC micelles (m) and PTXloaded TPGS/SPC/NaC micelles (g) in SGF (A) and in SIF (B) (n ¼ 3).

Figure 6. In vitro cytotoxicity of PTX-free F127-PEI/TPGS/SPC/NaC micelles and F127-PEI copolymers after incubation for 48 h with MCF7/Adr cells at different concentrations (n ¼ 3).

solution with verapamil resulted in an enhancement in plasma concentration and the absolute bioavailability of PTX was increased to 11.28%, due to the effect of verapamil against P-gp efflux pumps. Compared with PTX solution

To improve the stability and the oral bioavailability of SPC/NaC mixed micelle system, the stable micelles made of F127-PEI, TPGS, SPC and NaC were prepared. Through the electrostatic interaction and steric protection of long PEO segment in F127 molecule, the micelle aggregation became more tight and the stability was enhanced (Figure 1). Even though zeta-potential was increased to 1.5 ± 0.46 mV, the micelles still maintained integrity in aqueous solution. In addition, the modification of the micelles with Pluronic resulted in enhancing water solubility and reducing protein and enzyme adsorption, which is a critical important property for drug delivery systems. Both critical micelle concentration (CMC) and the storage stability test showed that F127-PEI/TPGS/SPC/NaC micelles were more stable than TPGS/SPC/NaC micelles, which confirmed the effects of electrostatic interaction between PEI and NaC and steric protection of the PEO segment on micelle stability. The stability test also revealed that both F127-PEI/TPGS/ SPC/NaC and TPGS/SPC/NaC micelles were stable in gastric juice. However, in SIF, the F127-PEI/TPGS/SPC/NaC micelles were more stable than TPGS/SPC/NaC micelles. This phenomenon could be contributed by the existence of pancreatin, a mixture of trypsinase, amylopsin and lipase which accelerated the degradation of TPGS/SPC/NaC materials in micelles. However, in the case of F127-PEI/TPGS/ SPC/NaC micelles, F127-PEI was incorporated into formulation and thicker hydrophilic shell was formed, which could protect micelles from the destruction of enzymes. Previous report also demonstrated lipid nanoparticles degraded or aggregated in intestinal medium. However, with a polyethylene glycol (PEG) coat, the stability of the suspension was improved [20]. Cytotoxicity of empty micelles Cytotoxicity is a major hurdle for clinical feasibility of polycationic carriers. Many researchers have reported that high-molecular-weight PEIs are more toxic than low-molecular-weight ones [26]. In addition, the toxicity of PEI is also related to the density of positive charge [27]. The previous research revealed that conjugation with poly (ethylene glycol) (PEG) and charge neutralizing with opposite charged entity

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Figure 7. Fluorescence images of MCF-7/Adr cells after incubation for 2 h with (a) Rh-123 solution, (b) Rh-123 solution with verapamil, (c) Rh-123-loaded SPC/NaC micelles, (d) Rh-123-loaded SPC/NaC micelles with verapamil (e) Rh-123-loaded TPGS/SPC/ NaC micelles, (f) Rh-123-loaded TPGS/SPC/ NaC micelles with verapamil, (g) Rh123-loaded F127-PEI/TPGS/SPC/NaC micelles and (h) Rh-123-loaded F127-PEI/ TPGS/SPC/NaC micelles with verapamil.

decreased cell toxicity of PEI [27]. Therefore, the lowmolecular-weight PEI (Mn ¼ 1800) was conjugated with F127 to design Pluronic-PEI polymer and further complexed with NaC. The obviously lower cytotoxicity of empty F127-PEI/ TPGS/SPC/NaC micelles than F127-PEI copolymer was observed, which confirmed that the conjugation with F127 and forming the F127-PEI/NaC complex decreased the cell toxicity of PEI. Uptake by resistant breast cancer cells Results of the fluorescence images demonstrated that the accumulation of Rh-123 in MCF-7/Adr cells in micelles was

increased compared with that of free Rh-123. More importantly, the accumulation of Rh-123 in F127-PEI/TPGS/SPC/ NaC and TPGS/SPC/NaC mixed micelles has shown a marked increase compared with Rh-123 in SPC/NaC mixed micelles without verapamil (Figures 7 and 8). As for rhodamine-123-loaded F127-PEI/TPGS/SPC/NaC micelles, however, there is only a slightly enhancement in the accumulation of Rh-123 in MCF-7/Adr cells compared with TPGS/SPC/NaC ones (Figures 7 and 8). The most evident explanation for these phenomena is that Rh-123-bearing micelles are capable of inhibition of P-gp efflux by TPGS. This property might be very important for the development of


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Figure 8. Flow cytometry analysis by MCF-7/Adr cells after incubation for 2 h with various Rh-123 formulations.

Table 2. Permeability rate constants (Ka) and effective permeability coefficient (Peff) of PTX from F127-PEI/TPGS/SPC/NaC micelles in the whole intestine at different concentration (Means ± SD, n ¼ 3). C (mg/mL) 25 50 100

Ka/min1 (103)

Peff/cmmin1 (102)

2.11 ± 0.86 2.20 ± 0.92 2.45 ± 0.71

4.30 ± 1.58 4.54 ± 1.70 4.89 ± 1.29

Table 3. Permeability rate constants (Ka) and effective permeability coefficient (Peff) of PTX from different formulations at the concentration of 50 mg/mL in the whole intestine in rats (Means ± SD, n ¼ 3).

Formulations PTX solution SPC/NaC micelles TPGS/SPC/NaC micelles F127-PEI/TPGS/SPC/NaC micelles

Ka/min1 (103)

Peff/cmmin1 (102)

0.18 ± 0.04 1.18 ± 0.23 2.21 ± 0.54 2.33 ± 0.77

0.32 ± 0.07 2.16 ± 0.41 4.38 ± 0.97 4.62 ± 1.41

micellar forms of poorly soluble drugs including those for oral administration. Permeability of PTX in micelles through rat intestine The permeability characteristics of PTX in F127-PEI/TPGS/ SPC/NaC micelles in the whole small intestine under low (25 mg/mL), middle (50 mg/mL) and high (100 mg/mL) concentrations were determined. The Ka and Peff exhibited approximate concentration-independent changes (Table 2), indicating that passive transfer by diffusion across lipid membranes was the major pathway for intestinal

permeability of PTX in F127-PEI/TPGS/SPC/NaC micelles. This phenomenon also implied that the free drug released from micelles and then diffused across lipid membranes. The unstirred water layer is one of the main barriers for the intestinal absorption of hydrophobic compounds. The permeability of PTX in SPC/NaC micelles was significantly higher than those of PTX in solution (p50.05; Table 3). This result was in agreement with the candesartan cilexetil-loaded lipid nanocarriers and confirmed that NaC could promote the drug across the unstirred water layer and transport drug to the endothelial lining [11]. Another important complication in application of many anticancer drugs such as PTX is the phenomenon of drug resistance in many cancer cells and in the gastrointestinal tract. Several investigators explained the poor bioavailability of PTX due to the multidrug efflux pump, P-gp that is abundantly present in the gastrointestinal tract. One may hypothesize that the oral bioavailability of anti-cancer drugs can be improved if the drug is administered with P-gp inhibiting drugs such as cyclosporine and verapamil analogs or P-gp inhibiting surfactants such as Pluronic or TPGS [28]. The permeabilities of PTX in TPGS/SPC/NaC and F127-PEI/ TPGS/SPC/NaC micelles were increased significantly (p50.05) compared with PTX in SPC/NaC micelles. However, slight enhancement was observed between PTXloaded F127-PEI/TPGS/SPC/NaC micelles and TPGS/SPC/ NaC ones. These results confirmed that drug resistance is an important factor limiting the PTX transport across the intestinal epithelium and were consistent with the results of uptake studies by cells.



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Figure 9. Plasma concentration–time curves of PTX in rats after intravenous administration of PTX injection at a dose of 4 mg/kg (A) and oral administration of PTX-loaded micelles at a dose of 20 mg/kg (B). (m) Oral administration of PTX-loaded F127-PEI/TPGS/SPC/NaC micelles; ( ) oral administration of PTX-loaded TPGS/SPC/NaC micelles; (D) oral administration of PTX injection and (g) oral administration of PTX injection with verapamil (n ¼ 5). Table 4. Pharmacokinetic parameters in rats after oral administration of various PTX formulations (20 mg/kg; Means ± SD, n ¼ 5).

Pharmacokinetic parameters AUC0–1 (mgh/L) MRT0–1 (h) Cmax (mg/L) Tmax (h) F (%)

PTX injectiona

PTX solution

PTX solution with verapamil

PTX-loaded TPGS/ SPC/NaC micelles

PTX-loaded F127-PEI/ TPGS/SPC/NaC micelles

1649 ± 129 2.786 ± 1.05 1389 ± 67.3 0.083

871 ± 79 14.11 ± 1.65 52 ± 4.8 1 10.56 ± 1.57

930 ± 94 11.40 ± 1.22 72 ± 8.6 4 11.28 ± 1.98

2387 ± 189 18.13 ± 2.86 131 ± 11.97 4 28.93 ± 2.23

3387 ± 204 10.09 ± 0.97 282 ± 20.34 6 41.15 ± 4.19

a

i.v. administration of PTX injection at a dose of 4 mg/kg.

The permeability of PTX in F127-PEI/TPGS/SPC/NaC micelles in various intestinal segments of rats demonstrated that the main segments of absorbed in intestine were duodenum and jejunum. Since there are not much Peyer’s patches in duodenum and jejunum, thus it may hypothesize that drug transportation through lymphoid aggregate was not the main absorption pattern by oral administration of PTX in F127-PEI/TPGS/SPC/NaC micelles. Pharmacokinetics studies in rats As shown in Figure 9, the oral absorption of PTX solution dissolved in a 50/50 (vol/vol) mixture of Cremophore EL/ dehydrated ethanol was extremely limited. While administration of PTX-loaded TPGS/SPC/NaC mixed micelles resulted in the enhancement on the absorption of PTX by oral administration, Cmax and AUC0 ! 1 of PTX in TPGS/SPC/ NaC mixed micelles were higher than those obtained with PTX solution. These results could be attributed to the roles of NaC and TPGS in forming materials, which promoted the drug across the unstirred water layer and inhibited the efflux mediated by intestinal P-gp, thereby promoting the permeation across the intestinal wall. More importantly, PTX in F127-PEI/TPGS/SPC/NaC micelles displayed highest AUC0–1 (3387 mgh/L) and Cmax (282 mg/L) and the absolute bioavailability was also increased to 41.15 ± 4.19%. This phenomenon could be explained by the positive zeta-potential of F127-PEI/TPGS/SPC/NaC

micelles, which could achieve an intimate contact with intestinal epithelial cells by electrostatic adherence and provided a steep concentration gradient at the permeability membrane. However, in the cell and permeability experiments, there were only slightly enhancements between F127-PEI/TPGS/SPC/NaC and TPGS/SPC/NaC micelles (Figures 7 and 8 and Table 3). This difference was perhaps related to the micelle stability. It may hypothesize that some parts of TPGS/SPC/NaC micelles disaggregated in intestine fluids because of enzymes and some drug released and precipitated in intestine fluids, which decreased the absorption of PTX by oral administration. In summary, the oral absorption of lipophilic drugs was mainly restrained by their solubilization in the gastrointestinal medium, diffusion across mucus and transport across the intestinal epithelium. These barriers could be effectively overcome after the insoluble drugs were incorporated into micelles. Our experimental results indicated that the absorption of PTX in intestine was largely increased by micelles. Incorporating NaC into micelles could improve the drug across the unstirred water layer and mucus and promote the intestinal absorption of PTX. Furthermore, some polymers such as Pluronic or TPGS have P-gp inhibiting properties, thus incorporating these polymers into formulation could enhance the transportation of drug across the intestinal epithelium and improve the oral absorption of drug. Finally, micelles should maintain integrity in intestine fluids until crossing the unstirred water layer and mucus, which is one of

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the main barriers for the absorption of hydrophobic compounds. In this respect, targeting micelles for the mucus layer may be a promising venue to prolong the duration of contact with the endothelial lining, thereby increasing the time for degradation and release.


Conclusions In this article, stable SPC/NaC mixed micelles were prepared with F127-PEI, TPGS, SPC and NaC to overcome the drawbacks of SPC/NaC micelle system as well as improve the intestinal absorption. The micelles exhibited nanometer range spherical structure with sustained release profile in in vitro. Permeability research in rat intestine indicated that passive transfer by diffusion across lipid membranes was the major pathway for intestinal permeability of PTX in F127-PEI/ TPGS/SPC/NaC micelles. The permeability of PTX through rat intestine could be improved by inhibition of the efflux mediated by intestinal P-gp and enhancement of the drug transportation across the unstirred water layer to the endothelial lining. Pharmacokinetic studies in rats also revealed improved oral bioavailability compared with PTX solution after oral administration. To sum up, F127-PEI/TPGS/SPC/ NaC mixed micelles were proved a promising drug delivery system for oral administration of PTX.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by Natural Science Foundation for Young Scholar of Shandong Province (ZR2013HQ011) and Scientific Research Foundation for Returned Scholars, Ministry of education of China.

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Improved oral absorption of cilostazol via sulfonate salt formation with mesylate and besylate.