http://informahealthcare.com/mnc ISSN: 0265-2048 (print), 1464-5246 (electronic) J Microencapsul, 2014; 31(8): 805–814 ! 2014 Informa UK Ltd. DOI: 10.3109/02652048.2014.940010
Folate-modified pluronic-polyethylenimine and cholic acid polyion complex micelles as targeted drug delivery system for paclitaxel Yimu Li, Yi Zhou, Bai De, and Lingbing Li
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Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan, China
Abstract
Keywords
The aim of the present study is to construct a type of polyion complex micelles made of PF127-PEI copolymer and cholic acid (CA) and to evaluate the potential of this type of micelles as a targeted drug delivery system for paclitaxel (PTX). To further improve the targeting capability of micelles, folate was also incorporated into micelles. The characteristics and antitumour activity in vitro were investigated. Enhanced solubility of PTX was achieved by incorporating into the micelles. The capability of the polyion complex micelles containing rhodamine 123 to increase the level of intracellular delivery was also observed using fluorescence microscopy. The cytotoxicity of PTX-loaded micelles against cancer cell in vitro was remarkably higher than that of free drug and was better when folate was incorporated into the micelles. These properties such as specificity towards the folate receptor and the low toxicity render folate-modified polyion complex micelles promising candidate for targeted PTX delivery.
Drug delivery system, folate modified, paclitaxel, pluronic-polycationic polymer, polyion complex micelles
Introduction Paclitaxel (PTX) has demonstrated significant anti-tumour activity in clinical trials against a broad range of solid tumours, including refractory ovarian cancer, metastatic breast cancer, non-small-cell lung cancer, AIDS-related Kaposi’s sarcoma, head and neck malignancies and other cancers (Wang et al., 2012). However, because of the poor aqueous solubility and low therapeutic index of PTX, the clinical application is extremely limited (Singla et al., 2002). To overcome these problems, several alternative pharmaceutical carriers have been developed for PTX delivery including liposomes (Zhao et al., 2011), polymeric micelles (Liang et al., 2011) and nanoparticles (Kollipara et al., 2010). Another significant obstacle for successful chemotherapy with PTX is multidrug resistance (MDR) in tumour cells. MDR is often found in many types of human tumours. It is often related to over-expression of drug efflux pumps, such as P-glycoprotein (P-gp) that dramatically reduces intracellular concentration of drugs (Li et al., 2010). Thus, there is a need for the development of alternate formulation of PTX to solve these problems. Pluronic block copolymers consist of ethylene oxide (EO) and propylene oxide (PO) blocks that are arranged in a basic EOx– POy–EOx structure. Pluronic block copolymers are benign and are widely used in a variety of FDA-approved pharmaceutical applications (Kabanov et al., 2002). These macromolecules are surface active and self-assemble into micelles above critical micelle concentrations (CMC) and certain temperatures. Interestingly, Pluronic unimers existing at concentrations below their CMC incorporate themselves into the cell membranes
Address for correspondence: Dr. Lingbing Li, Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. Tel: 86-531-88382015. Fax: 86-53188382548. E-mail: [email protected]
History Received 10 November 2013 Revised 24 June 2014 Accepted 25 June 2014 Published online 30 July 2014
thereby altering the membrane microviscosity and inhibiting functioning of some membrane proteins such as P-glycoprotein (P-gp) responsible for pumping chemotherapeutic drugs out of the cell and other key elements responsible for multidrug resistance in the tumourous cells (Zhang et al., 2011). Recently nanofabrication of polymer micelles was significantly advanced by employing charge driven self-assembly of block copolymers containing ionic and non-ionic blocks (‘‘block ionomers’’). During the last decades diverse materials were synthesised by reacting the block ionomers with oppositely charged molecules such as synthetic surfactants/lipids, proteins or DNA, etc. These materials can form micelle-like nanoparticles in aqueous solution, called ‘‘polyion complex micelles’’ (Bayo´Puxan et al., 2011; Li et al., 2012). From the drug delivery point of view, polyion complex micelles represent nanocarriers with the core–corona structure. Surrounded by a hydrophilic corona, the core is formed by the aggregates of surfactant molecules electrostatically bound to the polyion chain. This core can incorporate a variety of pharmaceutical agents through a combination of electrostatic, hydrophobic, and hydrogen bonding interactions. The size, structure, and loading capacity of the core can be altered by changing the ratio of the polyion and surfactant components in the mixture. In addition, such complex micelles display an ability to respond to the changes in environmental parameters, specifically, the temperature, added solutes, ionic strength, and pH. As a result these systems have a potential for a drug release triggered by the change in the environment within the targeted cell (Bayo´-Puxan et al., 2011; Li et al., 2012). Cholic acid (CA) is one of the major bile acids produced in the human liver. Its unique facial amphiphilicity makes it a very useful building block for synthesising biocompatible polymers for drug delivery. Oligomers of CA have been employed as functional molecular containers for caging hydrophobic molecules; however, their application in drug delivery is very limited mainly due to
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the small and unstable cavities and poor water solubility (Luo et al., 2010). The water soluble synthetic polycationic polymers have been employed in a variety of gene and drug delivery applications (Mecke et al., 2005; Hong et al., 2006). The generally accepted mechanism for internalisation of these polymers is polycationmediated endocytosis, a three-step process composed of binding with phospholipids and/or glycolipids in the membrane, internalisation into cells, and exit from the endosome (Mecke et al., 2005). Polyethylenimine (PEI) is one of the most efficient of the non-viral gene delivery vectors. The gene transfer efficiency of PEI is attributed to its ability to overcome lysosomal degradation, which is one of the main cellular barriers to effective gene transfer. However, the PEI has not shown significant therapeutic efficacy in vivo due to its rapid clearance from the circulation and accumulation within RES (reticuloendothelial system) sites (Ko et al., 2009). To solve this problem, in present work the Pluronic-polycationic polymers, Pluronic F127 and polyethylenimine (PEI) conjugates (PF127-PEI) were synthesised and polyion complex material made of PF127-PEI copolymer and CA (PF127-PEI/CA) was prepared through electrostatic interaction. In order to further improve the therapeutic index of micelles loading anti-cancer drugs, the ligand-mediated strategy has been applied. In this strategy, some ligands, whose receptors are expressed selectively or over expressed on tumour cells, are connected to the surface of the micelles. Folic acid (folate) is an attractive candidate molecule for targeting cancer cells because it is an essential vitamin for the biosynthesis of nucleotide bases and is consumed in elevated quantities by proliferating cells. The receptor for folic acid is over expressed in many human cancers including malignancies of the ovary, brain, kidney, breast, myeloid cells, and lung. Folate has been popularly employed as a targeting moiety of various anti-cancer agents to avoid their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells (Li et al., 2010). This work focused on the constructing a type of polyion complex micelles made of PF127-PEI and CA and evaluating the potential of this kind of micelles as a targeted drug delivery system for PTX. In aqueous solution PF127-PEI/CA could form polyion complex micelles with a hydrophobic core formed by neutralised CA as well as PPO and a hydrophilic shell formed by PEO. The number of CA in the micelle core is tunable and the loading capacity of the core can be altered by changing the ratio of the PF127-PEI and CA in the micelles. To further improve the targeting capability of micelles, Pluronic F127-folate copolymer (PF127-FA) was synthesised and incorporated into the micelle formulation. The structure, micelle formation, drug-loading properties, and in vitro cytotoxicity of the polyion complex micelles were characterised. Additionally, the capabilities of the polyion complex micelles and folate-modified polyion complex micelles containing rhodamine 123 to increase the level of intracellular delivery were studied using fluorescence microscopy and flow cytometry.
Materials and methods Materials PTX was purchased from Yunnan Hande Bio-Engineering Co. Ltd. (Yunnan, China). Pluronic F127 (PF127) was purchased from Sigma-Aldrich (St. Louis, MO) and used after additional purification. 1,1-Carbonyldiimidazole (CDI), polyethylenimine (PEI) (MW 2000), CA, folate and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC) were also purchased from Sigma-Aldrich (St. Louis, MO). All other solvents were of analytical or chromatographic grade.
J Microencapsul, 2014; 31(8): 805–814
Synthesis of PF127-PEI copolymers Preparation of CDI-activated Pluronic F127: CDI-activated Pluronic F127 was prepared using the reported method with modifications (Vinogradov et al., 2005). A total of 2.5 g (0.2 mmol) of purified Pluronic F127 was dissolved in dry tetrahydrofuran (THF) (30 mL). The solution was slowly added to a stirred solution of anhydrous THF containing 1,1carbonyldiimidazole (CDI, 48.62 mg, 0.30 mmol) in a drop-wise manner. The reaction was carried out for 6 h at room temperature with gentle stirring under nitrogen atmosphere. The activated Pluronic F127 was precipitated three times in ice-cold diethyl ether and dried under vacuum for 12 h, yield 85 wt. %. Synthesis of PF127-PEI copolymer: The PF127-PEI copolymer was synthesised using an emulsification/solvent evaporation method with modifications (Vinogradov et al., 2005). A dichloromethane solution (1 mL) containing 100 mg CDIactivated Pluronic F127 was added dropwise to an aqueous solution (10 mL, pH 9) of PEI (0.04% w/v). The mixture solution was sonicated in a Branson sonifier 450 for 3 min. The oilin-water emulsion solution was transferred to a rotary evaporator. Residual solvent was removed at 30 C until the solution became clear. After neutralising with hydrochloric acid, the solution was dialysed by a Spectra/Por dialysis membrane (Mw cutoff 50 000) against aqueous 0.01% ammonia solution. The structures of CDI-activated Pluronic F127 and PF127-PEI copolymer were confirmed by Fourier transform infrared (FT-IR). Dried samples were pressed with KBr powder into pellets. FT-IR spectra were obtained on FT-IR spectroscopy (Thermo Electron Scientific Instruments Corp., Fitchburg, WI). Synthesis of PF127-folate polymer (PF127-FA) PF127-FA copolymer was synthesised using a reported method with modifications (Lin et al., 2009). A total of 87.58 mg (0.20 mmol) of FA was dissolved in 3 mL of dried DMSO and added to a one neck flask. Then, 35.32 mg (0.22 mmol) of CDI was added, and the reaction was carried out at room temperature with gentle stirring in the dark for 1 d. A total of 0.62 g (0.05 mmol) of PF127 which has been previously dried overnight in vacuum was added to the above solution. The reaction was allowed to proceed in the dark for 1 d at room temperature. The reaction mixture was transferred into a dialysis tube (Spectra, Millipore, MWCO 1000, Billerica, MA) and dialysed for 3 d against double-deionised (DD) water, which was changed every 3–6 h. PF127-FA was recovered via lyophilisation. The resulting product was dried in a vacuum oven for 2 d, yielding 51% w/w of product. The product was stored in a dry box until use. The purified polymer samples were analysed by UV absorbance at 363 nm in PBS to measure the folate content. Preparation of drug-loaded micelles To prepare drug-loaded PF127-PEI/CA polyion complex micelles, 20 mg of drug and 100 mg of copolymers composed of different amounts of PF127-PEI and CA were dissolved in chloroform (4 mL) and the organic solvent was subsequently removed by rotary vacuum evaporation. The film formed was additionally freeze-dried in vacuum, hydrated with a suitable amount of 5mM HEPES-buffered saline (HBS). The resulting mixture was filtered through a 0.45-mm Nylon filter. The final samples were freeze dried and drug-loading content was determined. The drug-loaded PF127-PEI/PF127-FA/CA mixed micelles were also prepared by the method described above except that the materials were composed of the mixture of PF127-PEI and CA (100 mg, 80 wt %) and PF127-FA (11 mg, 10 wt %).
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DOI: 10.3109/02652048.2014.940010
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Determination of drug-loading content of micelles
FT-IR analysis
To evaluate the drug-loading content (DL) and loading efficiency (LE) of PTX in micelles, a predetermined amount of freeze-dried PTX-loaded micelles was dissolved in 5 mL of DMF after accurately weighted. The exact concentration of PTX in DMF was then determined by high-performance liquid chromatography (HPLC) according to a reported procedure with modifications (Wang et al., 2007). The HPLC analysis was carried out using an Agilent HP series 1200 HPLC system equipped with a G1314B Iso Pump and variable wavelength UV–VIS Detector (Santa Clara, CA). Twenty microlitres of each sample were injected at least three times into a Spherisorb ODS2 column, 4.6 250 mm (Analytical Cartridge Waters, Wexford, Ireland). The mobile phase was composed of acetonitrile-35 mM ammonium acetate buffer (50:45, v/v, pH 5) with a flow rate of 1.0 mL/ min. The UV absorption at 230 nm was measured. The calibration curve was constructed by determination of a series of DMF solutions containing empty micelle polymers and increasing amounts of PTX. The HPLC assay showed perfect linear in the scope of 0.2–40 mg/mL of PTX with a standard curve of A ¼ 48.69C–1.8153 (R2 ¼ 0.9996). The mean recovery of PTX was (101.1 ± 1.2) % (n ¼ 3). There was no interference of the empty micelles with the assay. The concentrations of PTX (C) were determined by comparing the peak areas (A) with the standard curve. DL content (%) and LE (%) were calculated by the following equations.
Functional group characterisation was carried out by FT-IR analysis using a FT-IR spectrometer (Thermo Electron Scientific Instruments Corp.). The samples were prepared by compressing the samples into pellets with potassium bromide, respectively.
DL% ¼
weight of drug in micelles 100 weight of micelles
LE% ¼
weight of drug in micelles 100 weight of drug added
Appearance and size distribution measurement The micelle size and size distribution were measured via dynamic laser light scattering particle size analyser (Zetasizer3000, Malvern Co., Malvern, UK) equipped with a He-Ne laser at a wavelength of 632 nm at a 90 detection angle. The concentration of the micelles was 10 mg/mL in a phosphate-buffered saline solution (pH 7.4) and the temperature was controlled. The measurement was carried out in triplicate for parallel contrast. The morphological features of the micelles were observed by transmission electron microscope (TEM) using a JEM-100CX electron microscope (JEOL, Tokyo, Japan). In a typical experiment, a drop of properly diluted micelle solution was placed on TEM grid, excess fluid was removed from the edge of the copper disk with a piece of filter paper. A drop of phosphotungstic acid (2 wt % aqueous solution) was immediately added to the grid and stained for 30 s. Zeta-potential measurements Micelle surface charge analysis was performed by Zeta-potential Analyser instrument (Zetasizer 3000, Malvern Co., Malvern, UK). The concentration of the micelles was 10 mg/mL in a phosphatebuffered saline solution (pH 6.0), and the temperature was controlled. The measurement was carried out in triplicate. Nuclear magnetic resonance spectroscopy The proton nuclear magnetic resonance (1H NMR) analysis was performed using1H NMR spectroscopy (Bruker 300 MHz AMX 300). All spectra were obtained at room temperature from 15% (wt/v) DMSO solutions.
In vitro release of drug-loaded micelles The in vitro release of PTX from micelles was evaluated by dialysis method using a dialysis bag with a molecular weight cutoff of 3000–5000 Da. The PTX-loaded micelle solution (2 mL) corresponding to 40 mg of PTX was added to the pre-swollen dialysis bag. Then the dialysis bag was tied and immersed into 20-mL phosphate buffer saline (PBS, pH 5.5 or 7.4) containing 1% w/v polysorbate 80 at 37 C with gentle stirring (Kamimura et al., 2012). At each given time interval, 2 ml of release medium was withdrawn and replaced with fresh release medium. The concentration of the released PTX was determined by HPLC method as described above. Serum stability of PTX-loaded PF127-PEI/PF127-FA/CA micelles In order to validate the stability of micelles, PTX-loaded micelles at the total polymer concentration of 5 mM were incubated with 1% bovine serum albumin (BSA) at 37 C for 72 h (Kamimura et al., 2012). Size distribution and encapsulation efficiency of the samples were studied as indexes after filtration by 0.22-mm nylon filter to remove insoluble PTX crystallised. In vitro cytotoxicity assay The anti-tumour activity of the drug-loaded micelles was evaluated by the MTT method (Kim et al., 2003). Human epithelial carcinoma cell line, HeLa cells were cultured in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were seeded at a density of 8 103 cells per well in 96-well plates and incubated for 24 h in a humidified atmosphere containing 5% CO2 at 37 C to allow cell attachment. The growth medium was then replaced with 200 mL medium containing increasing concentrations of each of the following substances: free PTX aqueous solution containing 50/50 (v/v) mixture of Cremophore EL (polyoxyl 35 castor oil)/dehydrated ethanol (ethanol 51%,v/v), PTX-loaded PF127-PEI/CA micelles and PTX-loaded PF127-PEI/PF127-FA/CA micelles. The cells were incubated for 72 h and washed three times with PBS. Then, 20 mL aliquots of MTT solution (5 mg/mL) were added into each well and the cells were incubated for an additional 4 h at 37 C. Thereafter, the medium was removed, and the cells were mixed with 200 mL of dimethyl sulfoxide (DMSO) to dissolve the MTT formazan crystals. The absorbance of each well was measured at 570 nm by an ELISA (Thermo Scientific, USA). Relative cell viability (R%) was calculated as follows: R% ¼
absorbancetest 100% absorbancecontrol
The IC50 was obtained using logistic model. Cell uptake test The cell uptake was evaluated by fluorescence microscopy (Yang et al., 2007). Rhodamine 123 (Rh-123), a fluorescent probe and a substrate for P-gp, was chosen as the model drug (Dabholkar et al., 2006). The Rh-123-loaded micelles were prepared using the same method with preparation of PTX-loaded micelles. Free Rh-123 was removed from the Rh-123-containing
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micelle dispersions via dialysis against DI water for 1 d. The Rh-123-loaded micelles were then filtered using disposable filters (0.22-mm pore size) for sterilisation. The different formulations of Rh-123: 5 mM of free Rh-123, PF127-PEI/CA micelles with Rh-123, PF127-PEI/PF127-FA/CA micelles with Rh-123 and PF127-PEI/PF127-FA/CA micelles with Rh-123 and 100 mM folic acid were evaluated on HeLa cells, respectively. HeLa cells were seeded in six-well plates at densities of approximately 400 000 per well and incubated using RPMI 1640 medium for 24 h. Subsequently, 1 mL of RPMI 1640 medium containing the Rh-123-loaded micelles or the Rh-123 solution was added into each well and incubated at 37 C for various time periods. At predetermined time intervals, cells were washed three times with ice-cold PBS, and then the fluorescence images of the samples were observed and obtained by a Nikon Eclipse E400 microscope (NIKON Corporation, Tokyo, Japan). For a quantitative study, after the rhodamine-123 unabsorbed were washed by cold PBS, 500 mL of trypsin PBS solution (2.5 mg/mL) was added into the plates. The cells were trypsinised for 1 min and then harvested by adding 2 mL complete culture medium. Intracellular uptake of rhodamine-123 in the cells was then detected using flow cytometry (FCM, Bio-Rad, Hercules, CA).
Results Preparation of PF127-PEI PF127-PEI copolymer was synthesised adopting emulsifying solvent evaporation method. Activated PF127 by CDI was conjugated with 1 amino at the end of PEI. The general synthesis of PF127-PEI copolymer is presented in Figure 1(A). Figure 2(A) shows the FT-IR spectra of PF127, PF127-PEI and PF127-FA copolymers. Compared with the spectrum of Pluronic F127, the spectrum of PF127-PEI clearly demonstrated the successful chemical conjugation since a characteristic peak of the carbonyl bond stretching appears at 1719.13 cm1 and NH
bond stretching appears at 3424.40 cm1. The 1H NMR spectra in DMSO-d6 were also used to study the chemical structure of PF127-PEI copolymer (Figure 2B) and showed the peaks at (ppm) ¼ 1.14 (d, 3H, CH3 of PPO), 3.41–3.62 (m, 3H, 4H, CH2CHO of PPO and CH2CH2O of PEO), 2.81 (CH2NH of PEI) (Figure 3). These results indicated that the PF127-PEI copolymer was synthesised. The intensities of the PEI protons (2.81 (CH2NH of PEI)) and methyl groups of PPO (1.14) were measured to calculate the degree of PEI substitution onto PF127 and estimate the molecular weight of polymer. The molecular weight of PF127-PEI was about 14 500 Da based on 1H NMR and GPC measurements. On the base of PF127 copolymer, the two hydroxyl groups were conjugated with the amino groups of FA under the catalysis of CDI, generating PF127-FA copolymer (Figure 1B). In the spectrum of PF127-FA (Figure 2A), a peak at 3423.99 cm1 was assigned to the OH group and the peak of 1631.75 cm1 was assigned to the characteristic peak of the amide group of PF127FA copolymer. The content of folate in PF127-FA copolymer was measured by a UV-vis spectrum and the result was 5.3wt %. Physicochemical characteristics of test micelles Figure 3 shows the TEM micrographs of the PF127-PEI/CA and PF127-PEI/PF127-FA/CA polyion complex micelles. The results showed that most of the drug-loaded micelles had a distinct spherical shape and smooth surface without any aggregation. Particle diameter of the micelles was measured using DLS. Results showed that the average diameters of micelles were smaller than 100 nm and size distributions exhibited a unimodal pattern. It is worth stressing that the average diameter was increased from 58.35 ± 6.59 to 65.89 ± 6.73 nm when the micelles were modified by folate. The drug-loading contents of folate-modified polyion complex micelles were measured by the HPLC method (Table 1). The results showed that the drug-loading content of folate-modified
Figure 1. Synthesis and structures of pluronic PF127-PEI copolymer (A) and Pluronic-FA copolymer (B).
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DOI: 10.3109/02652048.2014.940010
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Figure 2. FT-IR and 1H-NMR spectra of copolymers. (A) FT-IR spectra of PF127, PF127-PEI and PF127-FA copolymers; (B) 1H-NMR spectrum of PF127-PEI copolymer.
Figure 3. TEM images of PTX-loaded micelles. (A) PTX-loaded PF127-PEI/CA micelles; (B) PTX-loaded PF127-PEI/PF127-FA/CA micelles.
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Table 1. Characteristics and drug-loading content of the micelles (n ¼ 3). Formulation (molar ratio) [PF127-PEI/CA] DL/% EE/% Zeta potential (mV)
1:0.5
1:1
1:1.5
1:2
8.83 ± 0.89 60.2 ± 3.64 +6.51 ± 0.54
12.45 ± 1.65 68.8 ± 5.68 +3.38 ± 0.43
15.92 ± 1.89 84.84 ± 6.54 +0.52 ± 0.34
13.35 ± 1.47 75.33 ± 6.43 0.76 ± 0.87
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micelles was increased by increasing the CA ratio in micelles. When the ratio was 1:1.5 the optimal drug-loading content was obtained (15.9%). Meanwhile, at this ratio the zeta potential was approximately zero (+0.52 mV) (Table 1), which indicated that the PF127-PEI/CA complex was aggregated in the solution due to the charge neutralisation and the micelles were formed. In vitro release In vitro release of PTX from micelles was evaluated by dialysis method with polysorbate 80 (1%, w/v) included in release media. Polysorbate 80 (1%, w/v) was used as the solubiliser to guarantee pseudo-sink condition because the solubility of PTX could be increased prominently in release media with the presence of polysorbate 80 (Kamimura et al., 2012). Considering the initial PTX amount (40 mg) and the volume of media (20 mL) in this study, the media containing 1% Polysorbate 80 were qualified enough to provide a good sink condition for PTX release. The PTX release from F127-PEI/PF127-FA/CA micelles was performed and PTX solution was used as control. As shown in Figure 4, PTX was initially released rapidly and afterwards steadily from PF127-PEI/PF127-FA/CA micelles, which is consistent with the medication principle of anti-tumour drugs. More importantly, the release test also suggested that there existed a significant difference between the amounts of released PTX in media of pH 5.5 and pH 7.4. Under basic condition (pH 7.4) about 60% of initially loaded drug was released from PF127-PEI/PF127-FA/CA micelles within 12 h. However, in acidic condition (pH 5.5), about 80% of initially loaded drug was released within 12 h. This fact suggested that the PF127-PEI/PF127-FA/CA micelles had considerable pH sensitivity and the release rate of PTX relied on the environmental pH. Serum stability The serum stability test showed that the PTX-loaded PF127-PEI/ PF127-FA/CA micelles were stable in the presence of 1% FBS in the buffer and no changes in particle size and polydispersity of these micelles were determined for at least 72 h. The approximately 5.7% of initial amount of encapsulated PTX was released.
Figure 4. Paclitaxel release profile from PF127-PEI/PF127-FA/CA micelles (g) and PTX solution (m). (A) in pH 5.5 PBS buffer solution; (B) in pH 7.4 PBS buffer solution at 37 C. Data represented as mean ± S.D. (n ¼ 3).
In vitro cytotoxicity Initially, the cytotoxicities of empty PF127-PEI/PF127-FA/CA and PF127-PEI/PF127-FA micelles were investigated. The results showed that the empty PF127-PEI/PF127-FA/CA micelles had a slight cytotoxicity and the cytotoxicity was decreased with the reduction of micellar formulation concentration (Figure 5A). The anti-tumour activity of the PTX-loaded micelles was evaluated by the MTT method using human epithelial carcinoma cell line, HeLa cells. For comparison, the cytotoxicity of the free PTX was also evaluated (Figure 5B). The IC50 values for various formulations towards HeLa cells were 28.3 ± 3.3 mg/mL for free PTX, 6.32 ± 0.66 mg/mL for PTX-loaded PF127-PEI/CA micelles and 4.87 ± 0.58 mg/mL for PTX-loaded PF127-PEI/PF127-FA/CA micelles.
Cell uptake Results of the fluorescence images of HeLa cells after incubating with free Rh-123 and Rh-123-containing micelles at 37 C for distinct durations were shown in Figure 6. As is shown, after exposure of HeLa cells to free rhodamine 123, the accumulation of Rh-123 in cells was limited throughout the entire course of the study. On the contrary, for the Rh-123-containing PF127-PEI/CA and PF127-PEI/PF127-FA/CA micelles, the accumulation of Rh-123 in HeLa cells was increased compared with that of free Rh-123. The rank of the accumulation of Rh-123 was PF127-PEI/ PF127-FA/CA micelles 4 PF127-PEI/CA micelles 4 free PTX. And PF127-PEI/PF127-FA/CA micelles displayed highest accumulation of Rh-123 in HeLa cells.
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Figure 5. In vitro cytotoxicities of pure polymers. (A) (-˙- PF127-PEI/PF127-FA/CA complex; -g- PF127-PEI/PF127-FA copolymer) and PTX-loaded micelles (B) (-˙- free PTX solution; -g- PTX-loaded PF127-PEI/CA micelles; -m- PTX-loaded PF127-PEI/PF127-FA/CA micelles) (n ¼ 6).
For quantitative study, the fluorescence intensities of HeLa cells incubated with different formulations were assayed by flow cytometry and the results were listed in Table 2 and Figure 7.
micelles the loading content of PTX was about 3.35% w/w (Li et al., 2013).
Discussion
The PTX release from the PF127-PEI/PF127-FA/CA micelles was pH dependent with the release rate being much faster at acidic pH 5.5 than that at neutral pH 7.4. This phenomenon was probably due to the pKa value of CA (about pKa ¼ 6). At the neutral conditions the carboxylic acid groups were deprotonated and the micelles were stable. On the contrary, at the acidic pH conditions (pH5-6), the carboxylic acid groups were protonated, therefore, resulting in the micelle dissociation and accelerating PTX release from PF127-PEI/PF127-FA/CA micelles. Such release profile at lower pH is an important property for targeted drug delivery system. Because the pH in the endosome or lysosome is in the range of pH 5–6.
Development and characterisation of drug-loaded micelles The Pluronic–polycationic copolymer, PF127-PEI can associate into polyion complex micelles with CA in aqueous solution. The hydrophobic core is formed by neutralised CA as well as PPO and the hydrophilic shell is formed by PEO of Pluronic. The modification of the micelles with Pluronic results in enhancing water solubility and reducing immunogenicity of the carrier. It also protects the micelles in the biological milieu, prolonging their circulation time, which is a critical important property for drug delivery systems. In addition, through electrostatic interaction, the number of CA in the micelle core is tunable and the drug-loading content of the core can be altered by changing the ratio of the PF127-PEI and CA in the micelles. Appearance and size distribution measurement of micelles showed that the micelles were spherical in shape with a smooth surface and good dispersibility and micelle size was smaller than 100 nm, which indicated that stable, nano-sized micelles were formed. The PTX-loaded PF127-PEI/PF127-FA/CA micelles were stable in buffer solution containing 1% FBS for at least 72 h. This result was probably due to that the core of PTX-loaded PF127-PEI/PF127-FA/CA micelles was stabilised by both electrostatic and hydrophobic interactions. In addition the long hydrophilic shell formed by PEO was also a barrier with function of preventing degradation. Many studies have revealed that several factors affect the drugloading content and loading efficiency of micelles. Among them, the overriding factor is the polymer–drug compatibility. Thus, the drug-loading content can be improved by increasing the amount of core-forming materials. In the case of PF127-PEI/PF127-FA/ CA micelles, the loading content of PTX can be altered by changing the ratio of PF127-PEI and CA. The results demonstrated that the loading content of PTX in PF127-PEI/PF127-FA/ CA micelles was about 15.9% w/w, while in the normal PF127
In vitro release of drug-loaded micelles
In vitro cytotoxicity Cytotoxicity is a major hurdle for clinical feasibility of polycationic carriers. Many researchers have reported that highmolecular-weight PEIs are more toxic than low-molecular-weight ones (Guo et al., 2009). In addition, the previous research also revealed that conjugation of transferrin-PEI/DNA complexes with poly (ethylene glycol) (PEG) decreased cell toxicity of PEI (Godbey et al., 2001). Therefore, the low-molecular-weight PEI (Mn ¼ 1800) was conjugated with PF127 to design Pluronic– polycationic polymer to reduce the cytotoxicity of empty micelles. The results showed that when the PF127-PEI/PF127-FA/CA polymer concentration was below 240 mg/mL, the viability was about 90%. This observation confirmed that the conjugation with PF127 and forming the PF127-PEI/CA complex decreased the cell toxicity of PEI. The anti-tumour activity of the PTX-loaded micelles was evaluated by the MTT method using human cervical carcinoma cell line, HeLa cells. HeLa cells were chosen for this study because this cell line is known to express P-gp (Li et al., 2010). The results suggested that PTX-loaded micelles displayed higher cytotoxicity compared with that of the free drug, which could be explained by the enhanced solubility of PTX in the micelle solution and inhibition of P-gp efflux due to the presence of Pluronic in micelles.
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Figure 6. The fluorescence images of HeLa cells after incubating with free Rh-123 for 0.5 h (a); incubating with free Rh-123 for 1 h (b); incubating with Rh-123-loaded PF127PEI/CA micelles for 0.5 h (c); incubating with Rh-123-loaded PF127-PEI/CA micelles for 1 h (d); incubating with Rh-123-loaded PF127-PEI/PF127-FA/CA micelles for 0.5 h (e); incubating with Rh-123-loaded PF127PEI/PF127-FA/CA micelles for 1 h (f); incubating with Rh-123-loaded PF127-PEI/ PF127-FA/CA micelles plus 100 mM free folic acid for 0.5 h (g); incubating with Rh-123-loaded PF127-PEI/PF127-FA/CA micelles plus 100 mM free folic acid for 1 h.
Table 2. Percentage of Rh-123 taken up by HeLa cells after incubation for 2 h with various Rh-123 formulations (means ± SD, n ¼ 3).
Formulations Rh-123 solution Rh-123-load PF127-PEI/CA micelles Rh-123-load PF127-PEI/PF127-FA/CA micelles Rh-123-load PF127-PEI/PF127-FA/CA micelles + folate
Percentage of Rh-123 taken up in cells (%) 34.91 ± 0.63 88.94 ± 0.51 97.72 ± 0.72 89.73 ± 0.88
More interestingly, compared with non-modified micelles, the folate-modified PTX-loaded micelles demonstrated a superior cytotoxicity (Figure 5B). This difference implied that PTX in folate-modified micelles could be taken into HeLa cells better than that in no modified micelles due to the interaction between the folate on the micelle surface and the folate receptor on the
HeLa cell surface. It was the interaction between folate and folate receptors that ensured more drugs were pumped into the tumour cells and performed a better anti-cancer effect. Cell uptake Rh-123, a fluorescent probe, is a substrate for P-gp and can, therefore, be used as a marker for P-gp activity in cells (Dabholkar et al., 2006). As shown in Figure 6, for Rh-123-containing PF127PEI/CA micelles and folate-modified PF127-PEI/CA micelles, the accumulation of Rh-123 in HeLa cells was increased compared with that of free Rh-123. The most evident explanation for this phenomenon is that Rh-123-bearing micelles are capable of inhibition of P-gp efflux by Pluronic (Kabanov et al., 2002). This property might be very important for the development of micellar forms of poorly soluble anti-cancer drugs. More importantly, the Rh-123-containing PF127-PEI/PF127FA/CA micelles showed a marked increase of cellular accumulation compared with PF127-PEI/CA micelles (Figure 6), which evidently, because of the effective process of receptor-mediated
DOI: 10.3109/02652048.2014.940010
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Figure 7. Flow cytometry histograms of micelles internalised into HeLa cells for 2 h. (A) Rh 123 solution and PF127-PEI/CA micelles; (B) PF127-PEI/PF127-FA/CA and PF127-PEI/PF127-FA/CA micelles plus 100 mM free folic acid.
endocytosis and recycling of folate receptors after internalisation of micelle particles. This interaction was the reason why folate mediated micelles could decrease side effects, and at the same time they were able to enhance therapeutic effects. In order to evaluate the role of folate receptor in the cellular uptake of Rh-123 loaded PF127-PEI/PF127-FA/CA micelles, a competitive binding assay was performed. For these experiments, 100 mM of free folic acid was added to the cell culture wells. As shown in Figure 6, a significant reduction in cellular accumulation of Rh-123 was observed, suggesting that the Rh-123-containing PF127-PEI/PF127-FA/CA micelles might be endocytosed via the folate receptor. Moreover the fluorescence intensities of HeLa cells incubated with solutions above were assayed by flow cytometry. As shown in Table 2 and Figure 7, in comparison with free Rh-123, both PF127-PEI/CA and folate-modified PF127-PEI/CA micelles elevated the amount of the internalised Rh-123 and the folatemodified PF127-PEI/CA micelles displayed a highest degree of cellular internalised Rh-123. These results were consistent with the qualitative results by fluorescence chromatography and confirmed that folate-modified PF127-PEI/CA micelles have the ability to transport the drug into folate receptor overexpressed HeLa cells through the receptor-mediated endocytosis.
Conclusion Polyion complex material formed by PF127-PEI and CA was successfully prepared. These complexes spontaneously selfassemble in aqueous solution into micelle-like nanoparticles with average size of 50–70 nm. Moreover, folate-modified PF127PEI/CA micelles were also prepared and used for drug delivery of PTX. The drug-loading content of folate-modified PF127-PEI/CA micelles was significantly improved (15.9%) compared with PTX-loaded normal micelles (3.35%). The PTX release from the folate-modified PF127-PEI/CA micelles was pH dependent with the rate of the release being much faster at acidic pH 5.5 than that at neutral pH 7.4. PF127-PEI copolymer was full of amino groups and easy to be protonated. Thus PF127-PEI/CA micelles could enhance their uptake by phagocytic cells. The selectivity and targeting properties of micelles towards tumour cells expressing more folate receptors could also be improved by modifying folate on the surface of micelles. The cytotoxicity results confirmed that the in vitro anti-cancer activity of the drugs was improved when micelles were used as drug transport carriers. PTX-loaded PF127PEI/CA micelles exhibited superior anti-tumour activity compared with the PTX solution and folate mediated PF127-PEI/CA micelles exhibited better anti-tumour activity compared with the no modified micelles. These properties such as increasing solubility of PTX, pH dependent release, low toxicity and specificity towards the folate receptor render folate-modified
polyion complex micelles promising candidate for targeted PTX delivery.
Declaration of interest This work was supported by National Undergraduate Scientific and Technological Innovation Project of Shandong University (201310422089) and Natural Science Foundation for Young Scholar of Shandong Province (ZR2013HQ011). This article has not been published elsewhere and it has not been submitted simultaneously for publication elsewhere. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
References Bayo´-Puxan N, Dufresne MH, Felber AE, Castagner B, Leroux JC. Preparation of polyion complex micelles from poly (ethylene glycol)block-polyions. J Control Rel, 2011;156:118–27. Dabholkar RD, Sawant RM, Mongayt DA, Devarajan PV, Torchilin VP. Polyethylene glycol–phosphatidylethanolamine conjugate (PEG–PE)based mixed micelles: Some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux. Int J Pharm, 2006; 315(1–2):148–57. Godbey WT, Mikos AG. Recent progress in gene delivery using non-viral transfer complexes. J Control Rel, 2001;72:115–25. Guo QF, Shi S, Wang XH, Kan B, Gu YC, Shi XY, Luo F, Zhao X, Wei YQ, Qian ZY. Synthesis of a novel biodegradable poly (ester amine) (PEAs) copolymer based on low-molecular-weight polyethyleneimine for gene delivery. Int J Pharm, 2009;379:82–9. Hong S, Leroueil PR, Janus EK, Peters JL, Kober MM, Islam MT, Orr BG, Baker JR, Holl MMB. Interaction of polycationic polymers with supported lipid bilayers and cells: Nanoscale hole formation and enhanced membrane permeability. Bio Chem, 2006;17:728–34. Kabanov AV, Batrakova EV, Alakhov VY. Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J Control Rel, 2002;82(2–3):189–212. Kamimura M, Kim Jh, Kabanov AV, Bronich TK, Nagasaki Y. Block ionomer complexes of PEG-block-poly (4-vinylbenzylphosphonate) and cationic surfactants as highly stable, pH responsive drug delivery system. J Control Rel, 2012;160:486–94. Kim SY, Lee YM, Baik DJ, Kang JS. Toxic characteristics of methoxy poly (ethylene glycol)/poly ("-caprolactone) nanospheres; In vitro and in vivo studies in the normal mice. Biomaterials, 2003;24:55–63. Ko YT, Kale A, Hartner WC, Papahadjopoulos-Sternberg B, Torchilin VP. Self-assembling micelle-like nanoparticles based on phospholipid polyethyleneimine conjugates for systemic gene delivery. J Control Rel, 2009;133:132–8. Kollipara S, Bende G, Mowa S, Saha R. Application of rotatable central composite design in the preparation and optimization of poly(lacticco-glycolic acid) nanoparticles for controlled delivery of paclitaxel. Drug Dev Ind Pharm, 2010;36(11):1377–87. Li N, Li XR, Zhou YX, Li WJ, Zhao Y, Ma SJ, Li JW, Gao YJ, Liu Y, Wang XL, Yin DD. The use of polyion complex micelles to enhance the oral delivery of salmon calcitonin and transport mechanism across the intestinal epithelial barrier. Biomaterials, 2012;33: 8881–92.
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Y. Li et al.
Li N, Yang XG, Zhai GX, Li LB. Multifunctional pluronic/poly(ethylenimine) nanoparticles for anticancer drug. J Coll Interf Sci, 2010;350: 117–25. Li Y, Bi Y, Xi Y, Li LB. Enhancement on oral absorption of paclitaxel by multifunctional pluronic micelles. J Drug Targ, 2013; 21(2):188–99. Liang HJ, Yang Q, Deng L, Lu J, Chen J. Phospholipid–Tween 80 mixed micelles as an intravenous delivery carrier for paclitaxel. Drug Dev Ind Pharm, 2011;37(5):597–605. Lin JJ, Chen JS, Huang SJ, Ko JH, Wang YM, Chen TL, Wang LF. Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications. Biomaterials, 2009;30: 5114–24. Luo J, Xiao K, Li Y, Lee JS, Shi L, Tan YH, Xing Li, Cheng RH, Liu GY, Lam KS. Well-defined, size-tunable, multifunctional micelles for efficient paclitaxel delivery for cancer treatment. Bioconjugate Chem, 2010;21:1216–24. Mecke A, Majoros IJ, Patri AK, Baker JR, Holl MMB, Orr BG. Lipid bilayer disruption by polycationic polymers: The roles of size and chemical functional group. Langmuir, 2005;21:10348–54. Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm, 2002;235:179–92.
J Microencapsul, 2014; 31(8): 805–814
Vinogradov SV, Zeman AD, Batrakova EV, Kabanov AV. Polyplex nanogel formulations for drug delivery of cytotoxic nucleoside analogs. J Control Rel, 2005;107(1):143–57. Wang Y, Jiang G, Qiu T, Ding F. Preparation and evaluation of paclitaxelloaded nanoparticle incorporated with galactose-carrying polymer for hepatocyte targeted delivery. Drug Dev Ind Pharm, 2012; 38(9):1039–46. Wang YZ, Yu L, Han LM, Sha XY, Fang XL. Difunctional Pluronic copolymer micelles for paclitaxel delivery: Synergistic effect of folatemediated targeting and Pluronic-mediated overcoming multidrug resistance in tumor cell lines. Int J Pharm, 2007;337(1–2):63–73. Yang TF, Chen CN, Chen MC, Lai CH, Liang HF, Sung HW. Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle. Biomaterials, 2007;28:725–34. Zhang W, Shi Y, Chen Y, Ye J, Sha X, Fang X. Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials, 2011;32: 2894–906. Zhao L, Ye Y, Li J, Wei YM. Preparation and the in vivo evaluation of paclitaxel liposomes for lung targeting delivery in dogs. J Pharm Pharmacol, 2011;63:80–6.
Folate-modified pluronic-polyethylenimine and cholic acid polyion complex micelles as targeted drug delivery system for paclitaxel Yimu Li, Yi Zhou, Bai De, and Lingbing Li
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Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan, China
Abstract
Keywords
The aim of the present study is to construct a type of polyion complex micelles made of PF127-PEI copolymer and cholic acid (CA) and to evaluate the potential of this type of micelles as a targeted drug delivery system for paclitaxel (PTX). To further improve the targeting capability of micelles, folate was also incorporated into micelles. The characteristics and antitumour activity in vitro were investigated. Enhanced solubility of PTX was achieved by incorporating into the micelles. The capability of the polyion complex micelles containing rhodamine 123 to increase the level of intracellular delivery was also observed using fluorescence microscopy. The cytotoxicity of PTX-loaded micelles against cancer cell in vitro was remarkably higher than that of free drug and was better when folate was incorporated into the micelles. These properties such as specificity towards the folate receptor and the low toxicity render folate-modified polyion complex micelles promising candidate for targeted PTX delivery.
Drug delivery system, folate modified, paclitaxel, pluronic-polycationic polymer, polyion complex micelles
Introduction Paclitaxel (PTX) has demonstrated significant anti-tumour activity in clinical trials against a broad range of solid tumours, including refractory ovarian cancer, metastatic breast cancer, non-small-cell lung cancer, AIDS-related Kaposi’s sarcoma, head and neck malignancies and other cancers (Wang et al., 2012). However, because of the poor aqueous solubility and low therapeutic index of PTX, the clinical application is extremely limited (Singla et al., 2002). To overcome these problems, several alternative pharmaceutical carriers have been developed for PTX delivery including liposomes (Zhao et al., 2011), polymeric micelles (Liang et al., 2011) and nanoparticles (Kollipara et al., 2010). Another significant obstacle for successful chemotherapy with PTX is multidrug resistance (MDR) in tumour cells. MDR is often found in many types of human tumours. It is often related to over-expression of drug efflux pumps, such as P-glycoprotein (P-gp) that dramatically reduces intracellular concentration of drugs (Li et al., 2010). Thus, there is a need for the development of alternate formulation of PTX to solve these problems. Pluronic block copolymers consist of ethylene oxide (EO) and propylene oxide (PO) blocks that are arranged in a basic EOx– POy–EOx structure. Pluronic block copolymers are benign and are widely used in a variety of FDA-approved pharmaceutical applications (Kabanov et al., 2002). These macromolecules are surface active and self-assemble into micelles above critical micelle concentrations (CMC) and certain temperatures. Interestingly, Pluronic unimers existing at concentrations below their CMC incorporate themselves into the cell membranes
Address for correspondence: Dr. Lingbing Li, Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China. Tel: 86-531-88382015. Fax: 86-53188382548. E-mail: [email protected]
History Received 10 November 2013 Revised 24 June 2014 Accepted 25 June 2014 Published online 30 July 2014
thereby altering the membrane microviscosity and inhibiting functioning of some membrane proteins such as P-glycoprotein (P-gp) responsible for pumping chemotherapeutic drugs out of the cell and other key elements responsible for multidrug resistance in the tumourous cells (Zhang et al., 2011). Recently nanofabrication of polymer micelles was significantly advanced by employing charge driven self-assembly of block copolymers containing ionic and non-ionic blocks (‘‘block ionomers’’). During the last decades diverse materials were synthesised by reacting the block ionomers with oppositely charged molecules such as synthetic surfactants/lipids, proteins or DNA, etc. These materials can form micelle-like nanoparticles in aqueous solution, called ‘‘polyion complex micelles’’ (Bayo´Puxan et al., 2011; Li et al., 2012). From the drug delivery point of view, polyion complex micelles represent nanocarriers with the core–corona structure. Surrounded by a hydrophilic corona, the core is formed by the aggregates of surfactant molecules electrostatically bound to the polyion chain. This core can incorporate a variety of pharmaceutical agents through a combination of electrostatic, hydrophobic, and hydrogen bonding interactions. The size, structure, and loading capacity of the core can be altered by changing the ratio of the polyion and surfactant components in the mixture. In addition, such complex micelles display an ability to respond to the changes in environmental parameters, specifically, the temperature, added solutes, ionic strength, and pH. As a result these systems have a potential for a drug release triggered by the change in the environment within the targeted cell (Bayo´-Puxan et al., 2011; Li et al., 2012). Cholic acid (CA) is one of the major bile acids produced in the human liver. Its unique facial amphiphilicity makes it a very useful building block for synthesising biocompatible polymers for drug delivery. Oligomers of CA have been employed as functional molecular containers for caging hydrophobic molecules; however, their application in drug delivery is very limited mainly due to
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the small and unstable cavities and poor water solubility (Luo et al., 2010). The water soluble synthetic polycationic polymers have been employed in a variety of gene and drug delivery applications (Mecke et al., 2005; Hong et al., 2006). The generally accepted mechanism for internalisation of these polymers is polycationmediated endocytosis, a three-step process composed of binding with phospholipids and/or glycolipids in the membrane, internalisation into cells, and exit from the endosome (Mecke et al., 2005). Polyethylenimine (PEI) is one of the most efficient of the non-viral gene delivery vectors. The gene transfer efficiency of PEI is attributed to its ability to overcome lysosomal degradation, which is one of the main cellular barriers to effective gene transfer. However, the PEI has not shown significant therapeutic efficacy in vivo due to its rapid clearance from the circulation and accumulation within RES (reticuloendothelial system) sites (Ko et al., 2009). To solve this problem, in present work the Pluronic-polycationic polymers, Pluronic F127 and polyethylenimine (PEI) conjugates (PF127-PEI) were synthesised and polyion complex material made of PF127-PEI copolymer and CA (PF127-PEI/CA) was prepared through electrostatic interaction. In order to further improve the therapeutic index of micelles loading anti-cancer drugs, the ligand-mediated strategy has been applied. In this strategy, some ligands, whose receptors are expressed selectively or over expressed on tumour cells, are connected to the surface of the micelles. Folic acid (folate) is an attractive candidate molecule for targeting cancer cells because it is an essential vitamin for the biosynthesis of nucleotide bases and is consumed in elevated quantities by proliferating cells. The receptor for folic acid is over expressed in many human cancers including malignancies of the ovary, brain, kidney, breast, myeloid cells, and lung. Folate has been popularly employed as a targeting moiety of various anti-cancer agents to avoid their non-specific attacks on normal tissues as well as to increase their cellular uptake within target cells (Li et al., 2010). This work focused on the constructing a type of polyion complex micelles made of PF127-PEI and CA and evaluating the potential of this kind of micelles as a targeted drug delivery system for PTX. In aqueous solution PF127-PEI/CA could form polyion complex micelles with a hydrophobic core formed by neutralised CA as well as PPO and a hydrophilic shell formed by PEO. The number of CA in the micelle core is tunable and the loading capacity of the core can be altered by changing the ratio of the PF127-PEI and CA in the micelles. To further improve the targeting capability of micelles, Pluronic F127-folate copolymer (PF127-FA) was synthesised and incorporated into the micelle formulation. The structure, micelle formation, drug-loading properties, and in vitro cytotoxicity of the polyion complex micelles were characterised. Additionally, the capabilities of the polyion complex micelles and folate-modified polyion complex micelles containing rhodamine 123 to increase the level of intracellular delivery were studied using fluorescence microscopy and flow cytometry.
Materials and methods Materials PTX was purchased from Yunnan Hande Bio-Engineering Co. Ltd. (Yunnan, China). Pluronic F127 (PF127) was purchased from Sigma-Aldrich (St. Louis, MO) and used after additional purification. 1,1-Carbonyldiimidazole (CDI), polyethylenimine (PEI) (MW 2000), CA, folate and 1-(3-dimethylaminopropyl)-3ethylcarbodiimide hydrochloride (EDC) were also purchased from Sigma-Aldrich (St. Louis, MO). All other solvents were of analytical or chromatographic grade.
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Synthesis of PF127-PEI copolymers Preparation of CDI-activated Pluronic F127: CDI-activated Pluronic F127 was prepared using the reported method with modifications (Vinogradov et al., 2005). A total of 2.5 g (0.2 mmol) of purified Pluronic F127 was dissolved in dry tetrahydrofuran (THF) (30 mL). The solution was slowly added to a stirred solution of anhydrous THF containing 1,1carbonyldiimidazole (CDI, 48.62 mg, 0.30 mmol) in a drop-wise manner. The reaction was carried out for 6 h at room temperature with gentle stirring under nitrogen atmosphere. The activated Pluronic F127 was precipitated three times in ice-cold diethyl ether and dried under vacuum for 12 h, yield 85 wt. %. Synthesis of PF127-PEI copolymer: The PF127-PEI copolymer was synthesised using an emulsification/solvent evaporation method with modifications (Vinogradov et al., 2005). A dichloromethane solution (1 mL) containing 100 mg CDIactivated Pluronic F127 was added dropwise to an aqueous solution (10 mL, pH 9) of PEI (0.04% w/v). The mixture solution was sonicated in a Branson sonifier 450 for 3 min. The oilin-water emulsion solution was transferred to a rotary evaporator. Residual solvent was removed at 30 C until the solution became clear. After neutralising with hydrochloric acid, the solution was dialysed by a Spectra/Por dialysis membrane (Mw cutoff 50 000) against aqueous 0.01% ammonia solution. The structures of CDI-activated Pluronic F127 and PF127-PEI copolymer were confirmed by Fourier transform infrared (FT-IR). Dried samples were pressed with KBr powder into pellets. FT-IR spectra were obtained on FT-IR spectroscopy (Thermo Electron Scientific Instruments Corp., Fitchburg, WI). Synthesis of PF127-folate polymer (PF127-FA) PF127-FA copolymer was synthesised using a reported method with modifications (Lin et al., 2009). A total of 87.58 mg (0.20 mmol) of FA was dissolved in 3 mL of dried DMSO and added to a one neck flask. Then, 35.32 mg (0.22 mmol) of CDI was added, and the reaction was carried out at room temperature with gentle stirring in the dark for 1 d. A total of 0.62 g (0.05 mmol) of PF127 which has been previously dried overnight in vacuum was added to the above solution. The reaction was allowed to proceed in the dark for 1 d at room temperature. The reaction mixture was transferred into a dialysis tube (Spectra, Millipore, MWCO 1000, Billerica, MA) and dialysed for 3 d against double-deionised (DD) water, which was changed every 3–6 h. PF127-FA was recovered via lyophilisation. The resulting product was dried in a vacuum oven for 2 d, yielding 51% w/w of product. The product was stored in a dry box until use. The purified polymer samples were analysed by UV absorbance at 363 nm in PBS to measure the folate content. Preparation of drug-loaded micelles To prepare drug-loaded PF127-PEI/CA polyion complex micelles, 20 mg of drug and 100 mg of copolymers composed of different amounts of PF127-PEI and CA were dissolved in chloroform (4 mL) and the organic solvent was subsequently removed by rotary vacuum evaporation. The film formed was additionally freeze-dried in vacuum, hydrated with a suitable amount of 5mM HEPES-buffered saline (HBS). The resulting mixture was filtered through a 0.45-mm Nylon filter. The final samples were freeze dried and drug-loading content was determined. The drug-loaded PF127-PEI/PF127-FA/CA mixed micelles were also prepared by the method described above except that the materials were composed of the mixture of PF127-PEI and CA (100 mg, 80 wt %) and PF127-FA (11 mg, 10 wt %).
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DOI: 10.3109/02652048.2014.940010
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Determination of drug-loading content of micelles
FT-IR analysis
To evaluate the drug-loading content (DL) and loading efficiency (LE) of PTX in micelles, a predetermined amount of freeze-dried PTX-loaded micelles was dissolved in 5 mL of DMF after accurately weighted. The exact concentration of PTX in DMF was then determined by high-performance liquid chromatography (HPLC) according to a reported procedure with modifications (Wang et al., 2007). The HPLC analysis was carried out using an Agilent HP series 1200 HPLC system equipped with a G1314B Iso Pump and variable wavelength UV–VIS Detector (Santa Clara, CA). Twenty microlitres of each sample were injected at least three times into a Spherisorb ODS2 column, 4.6 250 mm (Analytical Cartridge Waters, Wexford, Ireland). The mobile phase was composed of acetonitrile-35 mM ammonium acetate buffer (50:45, v/v, pH 5) with a flow rate of 1.0 mL/ min. The UV absorption at 230 nm was measured. The calibration curve was constructed by determination of a series of DMF solutions containing empty micelle polymers and increasing amounts of PTX. The HPLC assay showed perfect linear in the scope of 0.2–40 mg/mL of PTX with a standard curve of A ¼ 48.69C–1.8153 (R2 ¼ 0.9996). The mean recovery of PTX was (101.1 ± 1.2) % (n ¼ 3). There was no interference of the empty micelles with the assay. The concentrations of PTX (C) were determined by comparing the peak areas (A) with the standard curve. DL content (%) and LE (%) were calculated by the following equations.
Functional group characterisation was carried out by FT-IR analysis using a FT-IR spectrometer (Thermo Electron Scientific Instruments Corp.). The samples were prepared by compressing the samples into pellets with potassium bromide, respectively.
DL% ¼
weight of drug in micelles 100 weight of micelles
LE% ¼
weight of drug in micelles 100 weight of drug added
Appearance and size distribution measurement The micelle size and size distribution were measured via dynamic laser light scattering particle size analyser (Zetasizer3000, Malvern Co., Malvern, UK) equipped with a He-Ne laser at a wavelength of 632 nm at a 90 detection angle. The concentration of the micelles was 10 mg/mL in a phosphate-buffered saline solution (pH 7.4) and the temperature was controlled. The measurement was carried out in triplicate for parallel contrast. The morphological features of the micelles were observed by transmission electron microscope (TEM) using a JEM-100CX electron microscope (JEOL, Tokyo, Japan). In a typical experiment, a drop of properly diluted micelle solution was placed on TEM grid, excess fluid was removed from the edge of the copper disk with a piece of filter paper. A drop of phosphotungstic acid (2 wt % aqueous solution) was immediately added to the grid and stained for 30 s. Zeta-potential measurements Micelle surface charge analysis was performed by Zeta-potential Analyser instrument (Zetasizer 3000, Malvern Co., Malvern, UK). The concentration of the micelles was 10 mg/mL in a phosphatebuffered saline solution (pH 6.0), and the temperature was controlled. The measurement was carried out in triplicate. Nuclear magnetic resonance spectroscopy The proton nuclear magnetic resonance (1H NMR) analysis was performed using1H NMR spectroscopy (Bruker 300 MHz AMX 300). All spectra were obtained at room temperature from 15% (wt/v) DMSO solutions.
In vitro release of drug-loaded micelles The in vitro release of PTX from micelles was evaluated by dialysis method using a dialysis bag with a molecular weight cutoff of 3000–5000 Da. The PTX-loaded micelle solution (2 mL) corresponding to 40 mg of PTX was added to the pre-swollen dialysis bag. Then the dialysis bag was tied and immersed into 20-mL phosphate buffer saline (PBS, pH 5.5 or 7.4) containing 1% w/v polysorbate 80 at 37 C with gentle stirring (Kamimura et al., 2012). At each given time interval, 2 ml of release medium was withdrawn and replaced with fresh release medium. The concentration of the released PTX was determined by HPLC method as described above. Serum stability of PTX-loaded PF127-PEI/PF127-FA/CA micelles In order to validate the stability of micelles, PTX-loaded micelles at the total polymer concentration of 5 mM were incubated with 1% bovine serum albumin (BSA) at 37 C for 72 h (Kamimura et al., 2012). Size distribution and encapsulation efficiency of the samples were studied as indexes after filtration by 0.22-mm nylon filter to remove insoluble PTX crystallised. In vitro cytotoxicity assay The anti-tumour activity of the drug-loaded micelles was evaluated by the MTT method (Kim et al., 2003). Human epithelial carcinoma cell line, HeLa cells were cultured in the RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin. The cells were seeded at a density of 8 103 cells per well in 96-well plates and incubated for 24 h in a humidified atmosphere containing 5% CO2 at 37 C to allow cell attachment. The growth medium was then replaced with 200 mL medium containing increasing concentrations of each of the following substances: free PTX aqueous solution containing 50/50 (v/v) mixture of Cremophore EL (polyoxyl 35 castor oil)/dehydrated ethanol (ethanol 51%,v/v), PTX-loaded PF127-PEI/CA micelles and PTX-loaded PF127-PEI/PF127-FA/CA micelles. The cells were incubated for 72 h and washed three times with PBS. Then, 20 mL aliquots of MTT solution (5 mg/mL) were added into each well and the cells were incubated for an additional 4 h at 37 C. Thereafter, the medium was removed, and the cells were mixed with 200 mL of dimethyl sulfoxide (DMSO) to dissolve the MTT formazan crystals. The absorbance of each well was measured at 570 nm by an ELISA (Thermo Scientific, USA). Relative cell viability (R%) was calculated as follows: R% ¼
absorbancetest 100% absorbancecontrol
The IC50 was obtained using logistic model. Cell uptake test The cell uptake was evaluated by fluorescence microscopy (Yang et al., 2007). Rhodamine 123 (Rh-123), a fluorescent probe and a substrate for P-gp, was chosen as the model drug (Dabholkar et al., 2006). The Rh-123-loaded micelles were prepared using the same method with preparation of PTX-loaded micelles. Free Rh-123 was removed from the Rh-123-containing
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micelle dispersions via dialysis against DI water for 1 d. The Rh-123-loaded micelles were then filtered using disposable filters (0.22-mm pore size) for sterilisation. The different formulations of Rh-123: 5 mM of free Rh-123, PF127-PEI/CA micelles with Rh-123, PF127-PEI/PF127-FA/CA micelles with Rh-123 and PF127-PEI/PF127-FA/CA micelles with Rh-123 and 100 mM folic acid were evaluated on HeLa cells, respectively. HeLa cells were seeded in six-well plates at densities of approximately 400 000 per well and incubated using RPMI 1640 medium for 24 h. Subsequently, 1 mL of RPMI 1640 medium containing the Rh-123-loaded micelles or the Rh-123 solution was added into each well and incubated at 37 C for various time periods. At predetermined time intervals, cells were washed three times with ice-cold PBS, and then the fluorescence images of the samples were observed and obtained by a Nikon Eclipse E400 microscope (NIKON Corporation, Tokyo, Japan). For a quantitative study, after the rhodamine-123 unabsorbed were washed by cold PBS, 500 mL of trypsin PBS solution (2.5 mg/mL) was added into the plates. The cells were trypsinised for 1 min and then harvested by adding 2 mL complete culture medium. Intracellular uptake of rhodamine-123 in the cells was then detected using flow cytometry (FCM, Bio-Rad, Hercules, CA).
Results Preparation of PF127-PEI PF127-PEI copolymer was synthesised adopting emulsifying solvent evaporation method. Activated PF127 by CDI was conjugated with 1 amino at the end of PEI. The general synthesis of PF127-PEI copolymer is presented in Figure 1(A). Figure 2(A) shows the FT-IR spectra of PF127, PF127-PEI and PF127-FA copolymers. Compared with the spectrum of Pluronic F127, the spectrum of PF127-PEI clearly demonstrated the successful chemical conjugation since a characteristic peak of the carbonyl bond stretching appears at 1719.13 cm1 and NH
bond stretching appears at 3424.40 cm1. The 1H NMR spectra in DMSO-d6 were also used to study the chemical structure of PF127-PEI copolymer (Figure 2B) and showed the peaks at (ppm) ¼ 1.14 (d, 3H, CH3 of PPO), 3.41–3.62 (m, 3H, 4H, CH2CHO of PPO and CH2CH2O of PEO), 2.81 (CH2NH of PEI) (Figure 3). These results indicated that the PF127-PEI copolymer was synthesised. The intensities of the PEI protons (2.81 (CH2NH of PEI)) and methyl groups of PPO (1.14) were measured to calculate the degree of PEI substitution onto PF127 and estimate the molecular weight of polymer. The molecular weight of PF127-PEI was about 14 500 Da based on 1H NMR and GPC measurements. On the base of PF127 copolymer, the two hydroxyl groups were conjugated with the amino groups of FA under the catalysis of CDI, generating PF127-FA copolymer (Figure 1B). In the spectrum of PF127-FA (Figure 2A), a peak at 3423.99 cm1 was assigned to the OH group and the peak of 1631.75 cm1 was assigned to the characteristic peak of the amide group of PF127FA copolymer. The content of folate in PF127-FA copolymer was measured by a UV-vis spectrum and the result was 5.3wt %. Physicochemical characteristics of test micelles Figure 3 shows the TEM micrographs of the PF127-PEI/CA and PF127-PEI/PF127-FA/CA polyion complex micelles. The results showed that most of the drug-loaded micelles had a distinct spherical shape and smooth surface without any aggregation. Particle diameter of the micelles was measured using DLS. Results showed that the average diameters of micelles were smaller than 100 nm and size distributions exhibited a unimodal pattern. It is worth stressing that the average diameter was increased from 58.35 ± 6.59 to 65.89 ± 6.73 nm when the micelles were modified by folate. The drug-loading contents of folate-modified polyion complex micelles were measured by the HPLC method (Table 1). The results showed that the drug-loading content of folate-modified
Figure 1. Synthesis and structures of pluronic PF127-PEI copolymer (A) and Pluronic-FA copolymer (B).
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Figure 2. FT-IR and 1H-NMR spectra of copolymers. (A) FT-IR spectra of PF127, PF127-PEI and PF127-FA copolymers; (B) 1H-NMR spectrum of PF127-PEI copolymer.
Figure 3. TEM images of PTX-loaded micelles. (A) PTX-loaded PF127-PEI/CA micelles; (B) PTX-loaded PF127-PEI/PF127-FA/CA micelles.
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Table 1. Characteristics and drug-loading content of the micelles (n ¼ 3). Formulation (molar ratio) [PF127-PEI/CA] DL/% EE/% Zeta potential (mV)
1:0.5
1:1
1:1.5
1:2
8.83 ± 0.89 60.2 ± 3.64 +6.51 ± 0.54
12.45 ± 1.65 68.8 ± 5.68 +3.38 ± 0.43
15.92 ± 1.89 84.84 ± 6.54 +0.52 ± 0.34
13.35 ± 1.47 75.33 ± 6.43 0.76 ± 0.87
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micelles was increased by increasing the CA ratio in micelles. When the ratio was 1:1.5 the optimal drug-loading content was obtained (15.9%). Meanwhile, at this ratio the zeta potential was approximately zero (+0.52 mV) (Table 1), which indicated that the PF127-PEI/CA complex was aggregated in the solution due to the charge neutralisation and the micelles were formed. In vitro release In vitro release of PTX from micelles was evaluated by dialysis method with polysorbate 80 (1%, w/v) included in release media. Polysorbate 80 (1%, w/v) was used as the solubiliser to guarantee pseudo-sink condition because the solubility of PTX could be increased prominently in release media with the presence of polysorbate 80 (Kamimura et al., 2012). Considering the initial PTX amount (40 mg) and the volume of media (20 mL) in this study, the media containing 1% Polysorbate 80 were qualified enough to provide a good sink condition for PTX release. The PTX release from F127-PEI/PF127-FA/CA micelles was performed and PTX solution was used as control. As shown in Figure 4, PTX was initially released rapidly and afterwards steadily from PF127-PEI/PF127-FA/CA micelles, which is consistent with the medication principle of anti-tumour drugs. More importantly, the release test also suggested that there existed a significant difference between the amounts of released PTX in media of pH 5.5 and pH 7.4. Under basic condition (pH 7.4) about 60% of initially loaded drug was released from PF127-PEI/PF127-FA/CA micelles within 12 h. However, in acidic condition (pH 5.5), about 80% of initially loaded drug was released within 12 h. This fact suggested that the PF127-PEI/PF127-FA/CA micelles had considerable pH sensitivity and the release rate of PTX relied on the environmental pH. Serum stability The serum stability test showed that the PTX-loaded PF127-PEI/ PF127-FA/CA micelles were stable in the presence of 1% FBS in the buffer and no changes in particle size and polydispersity of these micelles were determined for at least 72 h. The approximately 5.7% of initial amount of encapsulated PTX was released.
Figure 4. Paclitaxel release profile from PF127-PEI/PF127-FA/CA micelles (g) and PTX solution (m). (A) in pH 5.5 PBS buffer solution; (B) in pH 7.4 PBS buffer solution at 37 C. Data represented as mean ± S.D. (n ¼ 3).
In vitro cytotoxicity Initially, the cytotoxicities of empty PF127-PEI/PF127-FA/CA and PF127-PEI/PF127-FA micelles were investigated. The results showed that the empty PF127-PEI/PF127-FA/CA micelles had a slight cytotoxicity and the cytotoxicity was decreased with the reduction of micellar formulation concentration (Figure 5A). The anti-tumour activity of the PTX-loaded micelles was evaluated by the MTT method using human epithelial carcinoma cell line, HeLa cells. For comparison, the cytotoxicity of the free PTX was also evaluated (Figure 5B). The IC50 values for various formulations towards HeLa cells were 28.3 ± 3.3 mg/mL for free PTX, 6.32 ± 0.66 mg/mL for PTX-loaded PF127-PEI/CA micelles and 4.87 ± 0.58 mg/mL for PTX-loaded PF127-PEI/PF127-FA/CA micelles.
Cell uptake Results of the fluorescence images of HeLa cells after incubating with free Rh-123 and Rh-123-containing micelles at 37 C for distinct durations were shown in Figure 6. As is shown, after exposure of HeLa cells to free rhodamine 123, the accumulation of Rh-123 in cells was limited throughout the entire course of the study. On the contrary, for the Rh-123-containing PF127-PEI/CA and PF127-PEI/PF127-FA/CA micelles, the accumulation of Rh-123 in HeLa cells was increased compared with that of free Rh-123. The rank of the accumulation of Rh-123 was PF127-PEI/ PF127-FA/CA micelles 4 PF127-PEI/CA micelles 4 free PTX. And PF127-PEI/PF127-FA/CA micelles displayed highest accumulation of Rh-123 in HeLa cells.
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Figure 5. In vitro cytotoxicities of pure polymers. (A) (-˙- PF127-PEI/PF127-FA/CA complex; -g- PF127-PEI/PF127-FA copolymer) and PTX-loaded micelles (B) (-˙- free PTX solution; -g- PTX-loaded PF127-PEI/CA micelles; -m- PTX-loaded PF127-PEI/PF127-FA/CA micelles) (n ¼ 6).
For quantitative study, the fluorescence intensities of HeLa cells incubated with different formulations were assayed by flow cytometry and the results were listed in Table 2 and Figure 7.
micelles the loading content of PTX was about 3.35% w/w (Li et al., 2013).
Discussion
The PTX release from the PF127-PEI/PF127-FA/CA micelles was pH dependent with the release rate being much faster at acidic pH 5.5 than that at neutral pH 7.4. This phenomenon was probably due to the pKa value of CA (about pKa ¼ 6). At the neutral conditions the carboxylic acid groups were deprotonated and the micelles were stable. On the contrary, at the acidic pH conditions (pH5-6), the carboxylic acid groups were protonated, therefore, resulting in the micelle dissociation and accelerating PTX release from PF127-PEI/PF127-FA/CA micelles. Such release profile at lower pH is an important property for targeted drug delivery system. Because the pH in the endosome or lysosome is in the range of pH 5–6.
Development and characterisation of drug-loaded micelles The Pluronic–polycationic copolymer, PF127-PEI can associate into polyion complex micelles with CA in aqueous solution. The hydrophobic core is formed by neutralised CA as well as PPO and the hydrophilic shell is formed by PEO of Pluronic. The modification of the micelles with Pluronic results in enhancing water solubility and reducing immunogenicity of the carrier. It also protects the micelles in the biological milieu, prolonging their circulation time, which is a critical important property for drug delivery systems. In addition, through electrostatic interaction, the number of CA in the micelle core is tunable and the drug-loading content of the core can be altered by changing the ratio of the PF127-PEI and CA in the micelles. Appearance and size distribution measurement of micelles showed that the micelles were spherical in shape with a smooth surface and good dispersibility and micelle size was smaller than 100 nm, which indicated that stable, nano-sized micelles were formed. The PTX-loaded PF127-PEI/PF127-FA/CA micelles were stable in buffer solution containing 1% FBS for at least 72 h. This result was probably due to that the core of PTX-loaded PF127-PEI/PF127-FA/CA micelles was stabilised by both electrostatic and hydrophobic interactions. In addition the long hydrophilic shell formed by PEO was also a barrier with function of preventing degradation. Many studies have revealed that several factors affect the drugloading content and loading efficiency of micelles. Among them, the overriding factor is the polymer–drug compatibility. Thus, the drug-loading content can be improved by increasing the amount of core-forming materials. In the case of PF127-PEI/PF127-FA/ CA micelles, the loading content of PTX can be altered by changing the ratio of PF127-PEI and CA. The results demonstrated that the loading content of PTX in PF127-PEI/PF127-FA/ CA micelles was about 15.9% w/w, while in the normal PF127
In vitro release of drug-loaded micelles
In vitro cytotoxicity Cytotoxicity is a major hurdle for clinical feasibility of polycationic carriers. Many researchers have reported that highmolecular-weight PEIs are more toxic than low-molecular-weight ones (Guo et al., 2009). In addition, the previous research also revealed that conjugation of transferrin-PEI/DNA complexes with poly (ethylene glycol) (PEG) decreased cell toxicity of PEI (Godbey et al., 2001). Therefore, the low-molecular-weight PEI (Mn ¼ 1800) was conjugated with PF127 to design Pluronic– polycationic polymer to reduce the cytotoxicity of empty micelles. The results showed that when the PF127-PEI/PF127-FA/CA polymer concentration was below 240 mg/mL, the viability was about 90%. This observation confirmed that the conjugation with PF127 and forming the PF127-PEI/CA complex decreased the cell toxicity of PEI. The anti-tumour activity of the PTX-loaded micelles was evaluated by the MTT method using human cervical carcinoma cell line, HeLa cells. HeLa cells were chosen for this study because this cell line is known to express P-gp (Li et al., 2010). The results suggested that PTX-loaded micelles displayed higher cytotoxicity compared with that of the free drug, which could be explained by the enhanced solubility of PTX in the micelle solution and inhibition of P-gp efflux due to the presence of Pluronic in micelles.
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Figure 6. The fluorescence images of HeLa cells after incubating with free Rh-123 for 0.5 h (a); incubating with free Rh-123 for 1 h (b); incubating with Rh-123-loaded PF127PEI/CA micelles for 0.5 h (c); incubating with Rh-123-loaded PF127-PEI/CA micelles for 1 h (d); incubating with Rh-123-loaded PF127-PEI/PF127-FA/CA micelles for 0.5 h (e); incubating with Rh-123-loaded PF127PEI/PF127-FA/CA micelles for 1 h (f); incubating with Rh-123-loaded PF127-PEI/ PF127-FA/CA micelles plus 100 mM free folic acid for 0.5 h (g); incubating with Rh-123-loaded PF127-PEI/PF127-FA/CA micelles plus 100 mM free folic acid for 1 h.
Table 2. Percentage of Rh-123 taken up by HeLa cells after incubation for 2 h with various Rh-123 formulations (means ± SD, n ¼ 3).
Formulations Rh-123 solution Rh-123-load PF127-PEI/CA micelles Rh-123-load PF127-PEI/PF127-FA/CA micelles Rh-123-load PF127-PEI/PF127-FA/CA micelles + folate
Percentage of Rh-123 taken up in cells (%) 34.91 ± 0.63 88.94 ± 0.51 97.72 ± 0.72 89.73 ± 0.88
More interestingly, compared with non-modified micelles, the folate-modified PTX-loaded micelles demonstrated a superior cytotoxicity (Figure 5B). This difference implied that PTX in folate-modified micelles could be taken into HeLa cells better than that in no modified micelles due to the interaction between the folate on the micelle surface and the folate receptor on the
HeLa cell surface. It was the interaction between folate and folate receptors that ensured more drugs were pumped into the tumour cells and performed a better anti-cancer effect. Cell uptake Rh-123, a fluorescent probe, is a substrate for P-gp and can, therefore, be used as a marker for P-gp activity in cells (Dabholkar et al., 2006). As shown in Figure 6, for Rh-123-containing PF127PEI/CA micelles and folate-modified PF127-PEI/CA micelles, the accumulation of Rh-123 in HeLa cells was increased compared with that of free Rh-123. The most evident explanation for this phenomenon is that Rh-123-bearing micelles are capable of inhibition of P-gp efflux by Pluronic (Kabanov et al., 2002). This property might be very important for the development of micellar forms of poorly soluble anti-cancer drugs. More importantly, the Rh-123-containing PF127-PEI/PF127FA/CA micelles showed a marked increase of cellular accumulation compared with PF127-PEI/CA micelles (Figure 6), which evidently, because of the effective process of receptor-mediated
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Figure 7. Flow cytometry histograms of micelles internalised into HeLa cells for 2 h. (A) Rh 123 solution and PF127-PEI/CA micelles; (B) PF127-PEI/PF127-FA/CA and PF127-PEI/PF127-FA/CA micelles plus 100 mM free folic acid.
endocytosis and recycling of folate receptors after internalisation of micelle particles. This interaction was the reason why folate mediated micelles could decrease side effects, and at the same time they were able to enhance therapeutic effects. In order to evaluate the role of folate receptor in the cellular uptake of Rh-123 loaded PF127-PEI/PF127-FA/CA micelles, a competitive binding assay was performed. For these experiments, 100 mM of free folic acid was added to the cell culture wells. As shown in Figure 6, a significant reduction in cellular accumulation of Rh-123 was observed, suggesting that the Rh-123-containing PF127-PEI/PF127-FA/CA micelles might be endocytosed via the folate receptor. Moreover the fluorescence intensities of HeLa cells incubated with solutions above were assayed by flow cytometry. As shown in Table 2 and Figure 7, in comparison with free Rh-123, both PF127-PEI/CA and folate-modified PF127-PEI/CA micelles elevated the amount of the internalised Rh-123 and the folatemodified PF127-PEI/CA micelles displayed a highest degree of cellular internalised Rh-123. These results were consistent with the qualitative results by fluorescence chromatography and confirmed that folate-modified PF127-PEI/CA micelles have the ability to transport the drug into folate receptor overexpressed HeLa cells through the receptor-mediated endocytosis.
Conclusion Polyion complex material formed by PF127-PEI and CA was successfully prepared. These complexes spontaneously selfassemble in aqueous solution into micelle-like nanoparticles with average size of 50–70 nm. Moreover, folate-modified PF127PEI/CA micelles were also prepared and used for drug delivery of PTX. The drug-loading content of folate-modified PF127-PEI/CA micelles was significantly improved (15.9%) compared with PTX-loaded normal micelles (3.35%). The PTX release from the folate-modified PF127-PEI/CA micelles was pH dependent with the rate of the release being much faster at acidic pH 5.5 than that at neutral pH 7.4. PF127-PEI copolymer was full of amino groups and easy to be protonated. Thus PF127-PEI/CA micelles could enhance their uptake by phagocytic cells. The selectivity and targeting properties of micelles towards tumour cells expressing more folate receptors could also be improved by modifying folate on the surface of micelles. The cytotoxicity results confirmed that the in vitro anti-cancer activity of the drugs was improved when micelles were used as drug transport carriers. PTX-loaded PF127PEI/CA micelles exhibited superior anti-tumour activity compared with the PTX solution and folate mediated PF127-PEI/CA micelles exhibited better anti-tumour activity compared with the no modified micelles. These properties such as increasing solubility of PTX, pH dependent release, low toxicity and specificity towards the folate receptor render folate-modified
polyion complex micelles promising candidate for targeted PTX delivery.
Declaration of interest This work was supported by National Undergraduate Scientific and Technological Innovation Project of Shandong University (201310422089) and Natural Science Foundation for Young Scholar of Shandong Province (ZR2013HQ011). This article has not been published elsewhere and it has not been submitted simultaneously for publication elsewhere. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the article.
References Bayo´-Puxan N, Dufresne MH, Felber AE, Castagner B, Leroux JC. Preparation of polyion complex micelles from poly (ethylene glycol)block-polyions. J Control Rel, 2011;156:118–27. Dabholkar RD, Sawant RM, Mongayt DA, Devarajan PV, Torchilin VP. Polyethylene glycol–phosphatidylethanolamine conjugate (PEG–PE)based mixed micelles: Some properties, loading with paclitaxel, and modulation of P-glycoprotein-mediated efflux. Int J Pharm, 2006; 315(1–2):148–57. Godbey WT, Mikos AG. Recent progress in gene delivery using non-viral transfer complexes. J Control Rel, 2001;72:115–25. Guo QF, Shi S, Wang XH, Kan B, Gu YC, Shi XY, Luo F, Zhao X, Wei YQ, Qian ZY. Synthesis of a novel biodegradable poly (ester amine) (PEAs) copolymer based on low-molecular-weight polyethyleneimine for gene delivery. Int J Pharm, 2009;379:82–9. Hong S, Leroueil PR, Janus EK, Peters JL, Kober MM, Islam MT, Orr BG, Baker JR, Holl MMB. Interaction of polycationic polymers with supported lipid bilayers and cells: Nanoscale hole formation and enhanced membrane permeability. Bio Chem, 2006;17:728–34. Kabanov AV, Batrakova EV, Alakhov VY. Pluronic block copolymers as novel polymer therapeutics for drug and gene delivery. J Control Rel, 2002;82(2–3):189–212. Kamimura M, Kim Jh, Kabanov AV, Bronich TK, Nagasaki Y. Block ionomer complexes of PEG-block-poly (4-vinylbenzylphosphonate) and cationic surfactants as highly stable, pH responsive drug delivery system. J Control Rel, 2012;160:486–94. Kim SY, Lee YM, Baik DJ, Kang JS. Toxic characteristics of methoxy poly (ethylene glycol)/poly ("-caprolactone) nanospheres; In vitro and in vivo studies in the normal mice. Biomaterials, 2003;24:55–63. Ko YT, Kale A, Hartner WC, Papahadjopoulos-Sternberg B, Torchilin VP. Self-assembling micelle-like nanoparticles based on phospholipid polyethyleneimine conjugates for systemic gene delivery. J Control Rel, 2009;133:132–8. Kollipara S, Bende G, Mowa S, Saha R. Application of rotatable central composite design in the preparation and optimization of poly(lacticco-glycolic acid) nanoparticles for controlled delivery of paclitaxel. Drug Dev Ind Pharm, 2010;36(11):1377–87. Li N, Li XR, Zhou YX, Li WJ, Zhao Y, Ma SJ, Li JW, Gao YJ, Liu Y, Wang XL, Yin DD. The use of polyion complex micelles to enhance the oral delivery of salmon calcitonin and transport mechanism across the intestinal epithelial barrier. Biomaterials, 2012;33: 8881–92.
Journal of Microencapsulation Downloaded from informahealthcare.com by Dalhousie University on 11/09/14 For personal use only.
814
Y. Li et al.
Li N, Yang XG, Zhai GX, Li LB. Multifunctional pluronic/poly(ethylenimine) nanoparticles for anticancer drug. J Coll Interf Sci, 2010;350: 117–25. Li Y, Bi Y, Xi Y, Li LB. Enhancement on oral absorption of paclitaxel by multifunctional pluronic micelles. J Drug Targ, 2013; 21(2):188–99. Liang HJ, Yang Q, Deng L, Lu J, Chen J. Phospholipid–Tween 80 mixed micelles as an intravenous delivery carrier for paclitaxel. Drug Dev Ind Pharm, 2011;37(5):597–605. Lin JJ, Chen JS, Huang SJ, Ko JH, Wang YM, Chen TL, Wang LF. Folic acid–Pluronic F127 magnetic nanoparticle clusters for combined targeting, diagnosis, and therapy applications. Biomaterials, 2009;30: 5114–24. Luo J, Xiao K, Li Y, Lee JS, Shi L, Tan YH, Xing Li, Cheng RH, Liu GY, Lam KS. Well-defined, size-tunable, multifunctional micelles for efficient paclitaxel delivery for cancer treatment. Bioconjugate Chem, 2010;21:1216–24. Mecke A, Majoros IJ, Patri AK, Baker JR, Holl MMB, Orr BG. Lipid bilayer disruption by polycationic polymers: The roles of size and chemical functional group. Langmuir, 2005;21:10348–54. Singla AK, Garg A, Aggarwal D. Paclitaxel and its formulations. Int J Pharm, 2002;235:179–92.
J Microencapsul, 2014; 31(8): 805–814
Vinogradov SV, Zeman AD, Batrakova EV, Kabanov AV. Polyplex nanogel formulations for drug delivery of cytotoxic nucleoside analogs. J Control Rel, 2005;107(1):143–57. Wang Y, Jiang G, Qiu T, Ding F. Preparation and evaluation of paclitaxelloaded nanoparticle incorporated with galactose-carrying polymer for hepatocyte targeted delivery. Drug Dev Ind Pharm, 2012; 38(9):1039–46. Wang YZ, Yu L, Han LM, Sha XY, Fang XL. Difunctional Pluronic copolymer micelles for paclitaxel delivery: Synergistic effect of folatemediated targeting and Pluronic-mediated overcoming multidrug resistance in tumor cell lines. Int J Pharm, 2007;337(1–2):63–73. Yang TF, Chen CN, Chen MC, Lai CH, Liang HF, Sung HW. Shell-crosslinked Pluronic L121 micelles as a drug delivery vehicle. Biomaterials, 2007;28:725–34. Zhang W, Shi Y, Chen Y, Ye J, Sha X, Fang X. Multifunctional Pluronic P123/F127 mixed polymeric micelles loaded with paclitaxel for the treatment of multidrug resistant tumors. Biomaterials, 2011;32: 2894–906. Zhao L, Ye Y, Li J, Wei YM. Preparation and the in vivo evaluation of paclitaxel liposomes for lung targeting delivery in dogs. J Pharm Pharmacol, 2011;63:80–6.
