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Evaluation of polyethylene glycol-conjugated novel polymeric anti-tumor drug for cancer therapy
Colloids and Surfaces B: Biointerfaces 120 (2014) 168–175
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Evaluation of polyethylene glycol-conjugated novel polymeric anti-tumor drug for cancer therapy Joung-Pyo Nam 1 , Jun-Kyu Park 1 , Dong-Hee Son, Tae-Hun Kim, Sun-Jeong Park, Seong-Cheol Park, Changyong Choi, Mi-Kyeong Jang, Jae-Woon Nah ∗ Department of Polymer Science and Engineering, Sunchon National University, 255 Juang-ro, Suncheon 540-950, Jeollanam-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 January 2014 Received in revised form 3 April 2014 Accepted 19 April 2014 Available online 22 May 2014 Keywords: Paclitaxel PEG Transferrin Polymeric prodrugs
a b s t r a c t A novel polymeric prodrug (PXPEG) was prepared to enhance the solubility of an anti-cancer drug, paclitaxel, in aqueous solutions and decrease the cytotoxicity by PEGylation, which means PEG attached to another molecule. In addition, the targeting ligand, transferrin (TF), was modified to PXPEG to enhance the therapeutic efficacy. The targeting ligand-modified PXPEG (TFPXPEG) was examined by 1 H-NMR to confirm the successful synthesis. The synthesized TFPXPEG had better solubility than the free drug against aqueous solution. The particle size of TFPXPEG was approximately 197.2 nm and it had a spherical shape. The MTT assay showed that the anti-tumor efficiency of TFPXPEG was better than that of TF-unmodified PXPEG. In the KB tumor-bearing mouse model, the tumor volume of TFPXPEG treated groups was decreased dramatically by more than 2 fold or 3 fold compared to the PBS or PXPEG treated groups. The in vitro and in vivo evaluation showed that TFPXPEG had better efficacy than that of PXPEG due to the targeting effect of targeting ligands, such as TF. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is known medically as a malignant neoplasm and is comprised of a broad group of diseases involving uncontrolled cell growth. Cancer has genetic or environmental causes. Anand et al. reported that almost 90–95% of all cancer cases can be attributed to environmental factors, whereas the remaining 5–10% of all cancer cases can be attributed to genetic factors [1]. The number of cancer patients is increasing every year due to the changing environmental or genetic factors. Therefore, the number of deaths is also increasing. Many ways to overcome cancer have been reported. Paclitaxel (PX), a mitotic inhibitor, is one of the drugs most used for cancer therapy. PX is effective in clinical trials against a variety of tumors including breast cancer, Kaposi’s sarcoma, ovarian cancer, lung cancer [2–5]. On the other hand, its use should be limited to clinical trials because of the side effects including unusual bruising, pain/swelling/redness at the injection site, fever, cough, dizziness, shortness of breath, skin rash, and female infertility [2,6,7]. Most of these side effects are associated with the excipient used, such as Cremophor EL (CrmEL) and the polyoxyethylated castor oil. The
∗ Corresponding author. Tel.: +82 61 750 3566; fax: +82 61 750 5423. E-mail address: [email protected] (J.-W. Nah). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.04.013 0927-7765/© 2014 Elsevier B.V. All rights reserved.
commercially commonly used formulation, PX (Taxol® ), consists of CrmEL/ethanol (v/v, 50/50), which results in significant side effects [7,8]. Therefore, PX has two major problems when used clinically. One of the problems is the poor solubility and another problem is the side effects. In recent years, extensive studies have been reported to find a way of overcoming the major problems of PX (e.g. nanoparticles [9,10], liposome [11,12], polymeric micelles [13–16], emulsions [17], and polymeric prodrugs [18,19]). The use of polymeric prodrugs has many benefits, such as controlling the release of a drug, altering the drug biodistribution, altering the cellular uptake properties of a drug, taking advantage of the enhanced permeability and retention (EPR) effect, and prolonging the action of a drug [20]. Various bioavailable polymers have been used to prepare the polymeric prodrugs to overcome the disadvantages of the conventional drug. Polyethylene glycol (PEG) is one of the most widely used polymeric prodrugs system for the development of drugs. The structure of PEG is composed of the repeat elements (CH2 CH2 O). PEG imparts water solubility to hydrophobic drugs through PEGylation. PEGylation means PEG attached to another molecule, such a drug or therapeutic protein, via a covalent bond. The PEG moiety of a PEGylated polymeric prodrug has the following specific properties: high mobility in solution, water solubility, lack of toxicity and low immunogenicity, altered distribution in the body, and long circulation times in the body against the reducing renal clearance [21]. Veronese et al. reported
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that the PEG conjugated doxorubicin (anti-cancer agent) had a markedly prolonged plasma clearance and greater tumor targeting compared to free DOX [20]. Pawar et al. reported that PEG provided stability and enhanced aqueous solubility of the final PEGylated conjugate [22]. In this study, PEG-conjugated PX (PXPEG) was prepared to provide enhanced solubility and stability in aqueous solution. In addition, transferrin (TF), the targeting ligand, was conjugated to the side chain of PXPEG (TFPXPEG) for cancer therapy. TF can be bound to the transferrin receptors (TfR) present on the tumor cell surface, which leads to receptor mediated endocytosis. The surface of tumor cells overexpresses TfR because of the urgent requirement of iron to maintain cellular survival and shows 100 times more TfR than normal cells [23,24]. This targeting delivery system can provide lower systemic toxicity and higher therapeutics efficiency. This paper reports the design, synthesis, and in vitro and in vivo evaluation of TFPXPEG with TF as the targeting ligand to enhance the cellular uptake and anti-cancer activity through receptor-mediated endocytosis between TF and TfR. 2. Materials and methods 2.1. Materials The anti-cancer drug model, as a PX, was purchased from SigmaAldrich Chemical Co. (USA). TF, a targeting ligand (from human, 5% in H2 O, ≥98%, 80 kDa) was obtained from Sigma-Aldrich Chemical Co. (USA). COOH–polyethylene glycol–COOH (COOH–PEG–COOH, Mw : 3000 Da) was acquired from Sigma-Aldrich Chemical Co. (USA). Cross linkers, such as 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl) (DMAP), were supplied by SigmaAldrich Chemical Co. (USA). Dulbecco’s modified eagle medium (DMEM), Roswell park memorial institute (RPMI)-1640, trypsin, ethylenediaminetetraacetic acid (EDTA), and fetal bovine serum (FBS) were obtained from Gibco (BRL, MD, USA). All other chemicals and reagents were of extra reagent grade and used as received. 2.2. Methods 2.2.1. Synthesis of paclitaxel–polyethylene glycol polymeric prodrug (PXPEG) PXPEG was synthesized by a chemical reaction using the following method. A predetermined amount of PX (24 mg, 0.028 mmol) and PEG (168.64 mg, 2 equiv.) were dissolved in 1 mL dichloromethane. EDC (2.1 excess) and DMAP (2.1 excess) were then added to the PX and PEG dissolved solution. The reaction was carried out at room temperature for 24 h. After 24 h, the reaction was quenched with water and PXPEG was precipitated in excess diethyl ether. The powder of PXPEG was obtained by drying at 50 ◦ C. This powder was dissolved in distilled water and then it was dialyzed against distilled water for 3 d by using a molecular weight cut-off (MWCO, 3500 g/mol) dialysis membrane to remove the excess reagent. This solution was filtered through a 1.2 m pore sized syringe filter to remove large aggregates. Finally, the PXPEG polymeric prodrug was obtained as a white powder by lyophilization. 2.2.2. Targeting ligand-modified polymeric prodrug (TFPX–PEG) for targeting drug delivery Five milligram (1% of PXPEG, molar ratio) of TF was dissolved in 5 mL distilled water. EDC (2.1 excess) and NHS (2.1 excess) were then added to the TF dissolved solution at room temperature for 6 h with gentle stirring. 20 mg of PXPEG was dissolved in 5 mL of distilled water. A PXPEG solution and NHS-activated TF solution were mixed and reacted with gentle stirring at room temperature for 8 h.
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After the reaction, the resulting solution was introduced into the dialysis membrane (molecular weight cut off, MWCO 3500 Da) and dialyzed against distilled water to remove the unreacted materials, such EDC and NHS for 24 h. The total volume was adapted to 25 mL, and this solution was used to measure an in vitro and in vivo evaluation. In addition, the powder of TFPXPEG was obtained by lyophilization to examine the physicochemical properties. 2.2.3. Characterization of PXPEG and TFPXPEG polymeric prodrugs The polymeric prodrugs, such as PXPEG and TFPXPEG, were characterized by proton nuclear magnetic resonance spectroscopy (1 H-NMR) to confirm the successful synthesis. The1 H-NMR spectra of PXPEG and TFPXPEG were recorded on a 400 MHz NMR spectrometer (AVANCE 400FT-NMR 400 MHz, Bruker) using DMSO-d6 as the locking solvent. PX, PEG, PXPEG, TF, and TFPXPEG (5 mg of each) were dissolved in 0.5 mL of DMSO, and the products were assessed using an NMR spectrometer. The mean particle size and size distribution of the polymeric prodrugs were determined using an ELS-8000 electrophoretic LS spectrophotometer (NICOMP 380 ZLS zeta potential/particle sizer; Otsuka electronics Inc., Japan) equipped with a He–Ne laser beam at a wavelength of 632.8 nm at 25 ◦ C (scattering angle of 90◦ ). A sample solution (0.5 mg/mL) was used for the particle measurements (HE. 013.016-ALU cell adapter) without filtering. PXPEG and TFPXPEG (each 1 mg) was dissolved in 1 mL of deionized water to observe the morphology. Subsequently, 10 L of PXPEG and TFPXPEG suspended in deionized water were placed on a carbon film coated on a copper grid. The grid was stained with 1% uranyl acetate for 30 s and dried for 10 min. The polymeric prodrugs morphology was examined by transmission electron microscopy (TEM, JEOL JEM-2000 FX-II). The PX amounts of the PXPEG and TFPXPEG polymeric prodrugs were determined by high performance liquid chromatography (HPLC) [25]. The HPLC system consisted of a mobile phase delivery pump (LC-20AD HPLC pump, Shimadzu, Japan) and a UV detector (SPD-20A, UV/Vis detector, Shimadzu, Japan). A ZORBAX 300SB-C18 reverse-phase column (250 mm × 4.6 mm, 5 m, Agilent Technologies Inc., USA) was used to separate the components. The mobile phase was composed of acetonitrile and water at a ratio of 40:60 (v/v). The flow rate and column temperature were set to 1.0 mL/min and 30 ◦ C, respectively. The UV absorbance was determined to be 227 nm with an injection volume of 20 L. The amount of PX in PXPEG or TFPXPEG was calculated from standard curves. The assay was linear over the tested concentration range. The drug amount (DA) and grafting efficiency (GE) were calculated as follows: DA% =
weight of the drug in polymeric prodrug × 100% weight of the feeding polymer and drug
GE% =
weight of the drug in polymeric prodrug × 100% weight of the feeding drug
The solubility test of PXPEG and TFPXPEG against deionized water was examined using a UV spectrophotometer (VU1601, Shimazu, Japan). The concentrations of PX, PXPEG, and TFPXPEG were 1 mg/mL based on the amount of PX. 2.2.4. Cells and cell culture conditions The cell lines including human embryonic kidney 293 cells (HEK 293 cell) and cervical adenocarcinoma cells (KB cell) were purchased from KCLB® (Seoul, Republic of Korea). The cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified atmosphere containing 5% CO2 at 37 ◦ C. The cells grown as a monolayer were harvested by trypsinization (trypsin–EDTA).
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2.2.5. In vitro cytotoxicity and anticancer activity assay of polymeric prodrugs A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of PX, PXPEG, and TFPXPEG was performed to confirm the in vitro cell cytotoxicity and anticancer activity. The experiment was carried out as follows: HEK293 and KB cells were seeded into a 96-well micro plate at a density of 5 × 104 cells/well with 100 L. When the cell confluence reached 80%, the cells were treated with PX, PXPEG, and TFPXPEG for 48 h at equivalent concentrations ranging from 0.001 to 10 g/mL, which were based on the PX amount. After incubation, 10 L of the MTT solution (5 mg/mL in PBS) was added to each well, and the plate was incubated for an additional 2 h at 37 ◦ C. At a determined time, unreacted MTT was removed by aspiration. After removing the MTT-containing medium, the formazan crystals formed in the live cells were dissolved with 100 L DMSO. Finally, the absorbance was measured at 570 nm (optical density) and 670 nm (background subtraction) by VersaMax ELISA Microplate Reader StakMax® . The relative cell
viability (%) was calculated using the following equation [13]; OD value = (OD560 − OD670 )
Relative cell viability (%) =
ODsample − ODblank
(ODcontrol − ODblank )
× 100%
In addition, a TF competition assay was performed in KB cells to confirm the effect of the targeting ligand. The KB cells TFPXPEG were treated with a one-fold excess free TF and the absorbance was measured. 2.2.6. In vivo anticancer activity assay and H&E staining of polymeric prodrugs The xenografted tumors model of TfR overexpressed KB cells were established subcutaneously by injecting 1 × 107 cells into the abdomen of 6- to 8-week-old female athymic nude mice (Orientbio Inc.) to assess the antitumor effect of PXPEG or TFPXPEG. Once the size of the tumors reached 80–100 mm3 , the mice were divided randomly into three groups and injected intravenously with 100 L
Fig. 1. Scheme of novel polymeric prodrugs. (A) PEG conjugated to paclitaxel (PXPEG), (B) targeting ligand as a transferrin (TF) modified to PXPEG (TFPXPEG).
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PBS or PXPEG, or TFPXPEG three times at two day intervals (n = 5, 6.6 mg/kg). The anti-tumor efficacy was analyzed by measuring the size of the tumor using calipers three times weekly until the end of the study. The length and width of the tumor was measured, and the tumor volume was calculated using the following formula: tumor volume = 0.523Lw2 . For histological analysis, the tumor tissue was fixed in 4% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, embedded in paraffin, and cut into 5-m sections using a rotary microtome (Leica RM2125 RTS, © Leica biosystems, Germany). Representative sections were stained with hematoxylin (nucleus) and eosin (cytosol), and examined by optical microscopy. For immunohistochemistry, slides were deparaffinized in xylene and processed, as previously described [26]. The histological analysis was visualized by fluorescence microscopy.
stability in the body after a systemic injection. PEG was introduced to PX to obtain the PEG-grafted PX polymeric prodrug (PXPEG). PEG is the most widely used nonionic polymer because of its high aqueous solubility in the field of polymer-based drug delivery [21,27]. Di-carboxyl PEG (Mw : 3000 Da) was used to induce the hydrophobic drug and targeting ligand. PXPEG was synthesized by a coupling reaction between the carboxyl group of PEG and the hydroxyl group of PX. In addition, the targeting ligands, such as a TF, were introduced to PXPEG (TFPXPEG) via a coupling reaction. As shown in Fig. 1, the carboxymethyl group of PEG or PXPEG was activated by EDC and DMAP, which are known as a zero-crossing-linker because the amide bond was formed without leaving a spacer molecular [28]. The PX or TF was reacted with activated-PEG or activatedPXPEG to form the polymeric prodrug or target the polymeric prodrug.
2.2.7. Statistical analysis All data is reported as the mean ± standard deviation (SD) or standard error (SE) unless specified otherwise. The statistically significant differences were determined using Stat View software (Abacus Concepts, Inc., Berkeley, CA), and a Mann–Whitney test (non-parametric rank sum test). A P-value <0.05 was considered significant for evaluating the differences between the treatment groups (* P < 0.05; ** P < 0.01; *** P < 0.001).
3.2. Characterization of novel polymeric prodrugs
3. Result and discussion 3.1. Synthesis of novel polymeric prodrugs A novel polymeric prodrug was synthesized to overcome the low solubility of PX against deionized water and to enhance its
Fig. 2.
1
The formation of polymeric drugs, such as PXPEG and TFPXPEG was confirmed by 1 H-NMR spectroscopy using DMSO DMSO-d6 as the locking solvent. Figs. 2 and 3 show the proton peaks of PX, PEG, PXPEG, TF, and TFPXPEG. As shown in Fig. 2, the peak assignments of PX, PEG, and polymeric drug (PX conjugated to PEG) were classified as follows: PX (Fig. 1A), the proton peaks of CH3 were between 1.0 and 2.3 ppm, the proton peaks CH and CH2 were between 3.6 and 6.3 ppm, and the proton peaks CH of the aromatic group were between 7.2 and 9.0 ppm; COOH–PEG–COOH (Fig. 1B), the proton peaks of CH2 were observed at 3.5 ppm; PXPEG (Fig. 1C), the proton peaks of PX and PEG were confirmed and were chemically shifted due to the steric effect. The shifted peak assignments are as follows: CH2 peak (non-repeat molecular) of PEG, 2.6 and 3.2 ppm
H-NMR spectra of paclitaxel (A), COOH–PEG–COOH (B), and PXPEG (C).
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Fig. 3.
1
H-NMR spectra of PXPEG (A), TF (B), and TFPXPEG (C).
from 3.5 ppm; CH peak of PX (in aromatic group), and 6.8–9.3 ppm from 7.2 to 9.0 ppm. In addition, the peak assignments of TP and TP-modified PXPEG were as follows: TP (Fig. 3B), 0.8–1.3 ppm; TFPXPEG (Fig. 3C), the proton peaks of TF and PXPEG were chemically shifted due to the steric effect. CH2 (non-repeat molecular of PEG) was shifted from 2.6 and 3.2 ppm to 3.5 ppm because the macromolecule as a TF was introduced to the carboxyl group of PEG in PXPEG. 1 H-NMR showed that PXPEG and TFPXPEG were synthesized successfully. The synthesized polymeric prodrugs, such as PXPEG and TFPXPEG, have amphiphilic characteristics due to the hydrophilic portion as a PEG and hydrophobic portion as a PX. These amphiphilic characteristics can form particles such as a micelle by self-aggregation. The micelles were formed by self-aggregation and consisted of a hydrophobic core and hydrophilic shell in an aqueous environment. Therefore, many studies using polymeric micelles for tumor therapeutics have been reported [16,29–31]. As shown in Fig. 4, the particle size distribution (Fig. 4A) and morphology (Fig. 4B) of PXPEG or TFPXPEG were indicated. The particle size distribution and morphology were measured by dynamic light scattering (DLS) and TEM. PXPEG and TFPXPEG showed a unimodal
and narrow particle size distribution (Fig. 4A (a and b)). PXPEG and TFPXPEG had a spherical shape. Table 1 lists the particle size, feed composition, drug amount (DA), grafting efficiency, and the yield of the polymeric prodrugs. When TF was conjugated to PXPEG, the particle size was increased from 127.2 ± 87.3 to 197.2 ± 56.3. The increasing particle size of the polymeric prodrugs was observed by both DLS and TEM. In addition, TF was conjugated successfully to PXPEG and was present on the surface of PXPEG but not inside. The presence of a targeting ligand on the drug carrier can enhance the cellular uptake by receptor-mediated endocytosis [13,22,32]. The DAs of PXPEG and TFPXPEG calculated by HPLC were 11.8% and 10.6%. In particular, the GEs of PXPEG and TFPXPEG were 53.3% and 47.9%, respectively. The yield was less than 50%. The reason for the low yield suggests that the final products were filtered through a 1.2 m pore sized syringe filter to remove the large aggregates. 3.3. Solubility test against aqueous solution Generally, PX has very poor solubility in aqueous solutions; the solubility of PX in water is 0.0003 M. Therefore, the use of PX can cause side effects due to the excipient, such as Cremophor EL
Table 1 Feed composition and characterization of polymeric prodrugs (n = 3). Sample
Amount of PX (mg)
Amount of PEG (mg)
Amount of TF (mg)
DAa (%)
GEb (%)
Yieldc (%)
Particle sized (nm)
PXPEG TFPXPEG
24 24
84.32 84.32
– 5
11.8 10.6
53.3 47.9
41.5 40.3
127.2 ± 87.3 197.2 ± 56.3
a b c d
Degree of drug amount in polymeric prodrugs, calculated by HPLC. Degree of grafting efficiency to PEG, calculated by HPLC. Weight of final product/weight of initial polymer and drug × 100. Determined by DLS measurement.
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Fig. 4. Particle size and morphological observation of polymeric prodrugs. (A) Particle size of PXPEG (a) and TFPXPEG (b) analyzed by DLS. (B) Spherical forms of PXPEG (a) and TFPXPEG (b) were observed by using transmission electron microscope.
(CrmEL) [7,8,14]. In this study, to overcome the poor solubility of PX in aqueous solutions, the polymeric prodrugs were prepared with PEG and TF, which are hydrophilic molecules. This resulted in an increase in the solubility of PX (Fig. 5). The concentration of
each sample (PX, PXPEG, and TFPXPEG) was based on the amount of PX (final PX concentration: 1 mg/mL). The transmittances of PX, PXPEG, and TFPXPEG were 0.61%, 90.10%, and 97.70% at 400 nm, respectively. Interestingly, the hydrophilic molecular-introduced PXPEG and TFPXPEG showed a significant increase in solubility whereas PX was opaque. In addition, TF-conjugated polymeric prodrug (TFPXPEG) showed higher solubility than the other samples, such as PX and PXPEG. This shows that the hydrophilicity of PX was increased due to the introduction of hydrophilic molecules, such as PEG and TF. This shows that the prepared novel polymeric prodrugs can increase the solubility of PX. Forrest et al., reported that the paclitaxel prodrugs prepared by poly(ethylene glycol)-bpoly(-caprolactone) showed high solubility [19]. Lee et al., also reported that hydrophilic molecular (hyaluronic acid (HA) and PEG)-conjugated paclitaxel showed increased solubility [33].
3.4. In vitro cytotoxicity and anti-tumor activity of polymeric prodrugs
Fig. 5. Solubility photography of PX, PXPEG, and TFPXPEG against aqueous solution and transmittance was measured using UV.
A MTT assay was used to measure the cytotoxicity and antitumor activity of the polymeric prodrugs. The normal cells and cancer cells were treated with various formulations of PX, PXPEG, and TFPXPEG polymeric prodrugs at concentrations ranging from 0.001 to 10 g/mL, which are based on the PX amount. As shown in Fig. 6A, the cytotoxicity was examined on the normal cell as HEK293 cells, the cytotoxicity of the PEG and PX-introduced polymeric prodrugs (PXPEG and TFPXPEG) was reduced significantly compared to free PX. The viability of the cells treated with PXPEG and TFPXPEG was more than 60% at all concentrations, whereas the cell viability of PX was less than 60%. In particular, the cell viability of PXPEG and
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Fig. 6. (A) Cytotoxicity of polymeric prodrugs against HEK 293 cells and (B) anti-tumor efficiency of polymeric prodrugs against KB cells in vitro by MTT assay. Each data point represents the mean ± standard deviation (n = 3).
TFPXPEG was 62.2% and 72.8% at high concentrations (10 g/mL), compared to PX (21.2%). In addition, TFPXPEG showed the highest cell viability. The MTT assay showed that PEG and TF decreased the cytotoxicity of PX. As shown in Fig. 6B, the anti-tumor activity of the polymeric prodrugs on the cancer cell line, KB cell, was examined. The cell viability of PXPEG and TFPXPEG decreased with increasing PX concentration, and there were no significant differences between the two drugs. At the lowest concentration (0.001 g/mL), PXPEG and TFPXPEG exhibited efficient anti-tumor effects. In particular, TFPXPEG showed the highest anti-tumor efficiency at all concentrations. This suggests that the TFPXPEG uptake by the cells is higher than PXPEG due to receptor-mediated endocytosis. A previous study reported that TF modified micelles effectively targeted the TfR present on the surface of the cancer cells [13]. Bellocq et al. reported that TF-modified cyclodextrin polymer showed increased transfection efficiency due to the tumor-targeting ability of TF [34]. Fig. 6B shows the results of the competition assay. The TFPXPEG-treated cells after incubation with free TF showed lowest anti-tumor efficiency. The decreased anti-tumor efficiency of TFPXPEG (incubated with free TF) can be explained because of the lower number of binding sites (TfR) that can bind to TF. Zhang et al. also reported that the binding of human TF in Caco-2 cells was apparently inhibited by the addition of a one-fold excess of free TF [35]. The lowest anti-tumor efficiency of TFPXPEG (incubated with free TF) clearly showed TFPXPEG uptake into the cells by endocytosis between TF and TfR. Interestingly, despite the lower number of binding sites on the tumor cells surface, when the dose was increased, TFPXPEG (incubated with free TF) showed similar anti-tumor efficiency to PXPEG. The reason for this is that TFPXPEG has two types of targeted drug delivery: active targeted drug delivery, such as receptor-mediated endocytosis; and passive targeted drug delivery, such as the EPReffect.
PBS, PXPEG, and TFPXPEG, respectively. Similar to the MTT assay, the tumor volume of the PXPEG or TFPXPEG treated group was compared with the PBS treated group. In addition, TF-modified TFPXPEG showed higher effective therapeutic efficacy, more than 2 fold or 3 fold, compared to PXPEG (P < 0.05) or PBS (P < 0.05). This shows that targeting the ligand-modified TFPXPEG is more effective than PXPEG in terms of tumor inhibition due to the active targeting effect. In addition, the histological observations of PXPEG and TFPXPEG polymeric prodrug-treated tumor tissues were carried out after H&E staining to confirm the therapeutic efficacy of the polymeric prodrugs in the KB tumor-bearing mice (Fig. 7B). These histological
3.5. In vivo anti-tumor effect and immunohistological analysis of polymeric prodrugs in KB tumor-bearing mice To evaluate the anti-tumor effects of the PXPEG and TFPXPEG polymeric drugs, the tumor growth rate was examined after an intravascular injection (i.v. injection) of polymeric drugs into the KB tumor-bearing mice (Fig. 7A). The tumor was induced by a subcutaneous injection of KB cells, and PBS, PXPEG and TFPXPEG were injected once the size of the tumors reached 80–100 mm3 in volume. After twenty days from the first treatment day, the mean tumor volume was 2468.3 mm3 , 1742.3 mm3 , and 792.3 mm3 for
Fig. 7. In vivo anti-tumor efficiency and histological analysis of polymeric prodrugs in KB tumor subcutaneous tumor models (n = 5). (A) Inhibition of tumor growth by intravenous injection of PXPEG or TFPXPEG polymeric prodrugs (6.6 mg/kg). * P < 0.05 vs PBS treated group; * P < 0.05 vs PXPEG treated group. (B) Representative photographs of hematoxylin and eosin (H&E) staining in tumor tissues. Original magnification: ×400.
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differences were quantified by the morphometric determination of the proportion of the total tumor area that was necrotic in the H&E staining section [36]. As shown in Fig. 7B, the cells of the PBS-treated group were healthy with a high density. In contrast, the cells in the PXPEG or TFPXPEG polymeric prodrug-treated group showed induced necrosis, and the necrotic area of the tumors treated with the PXPEG or TFPXPEG polymeric prodrugs increased dramatically. In particular, the TFPXPEG treated group showed the largest increase in necrotic area. The MTT assay, tumor volume, and histological observation clearly showed that TFPXPEG can inhibit tumor growth. 4. Conclusion Novel polymeric prodrugs (TFPXPEG) with targeting ligand were prepared. TFPXPEG showed high solubility in an aqueous solution and formed nanoparticles at approximately 197.2 nm. The cytotoxicity of drug decreased when modified by PEG and TF to PXPEG. In addition, the anti-tumor efficiency of the polymeric prodrugs was effective against KB cells in vitro and in vivo. The in vivo and in vitro evaluation clearly showed that the targeting ligand, TF, which was modified to PXPEG, showed therapeutic efficacy and tumor growth inhibition through receptor-mediated endocytosis between TF of TFPXPEG and TfR present on the KB cell surface. Therefore, this study provides a novel polymeric drug for extending the utilization of the PEG-based drug delivery system as well as possible high tumor targeting between the targeting ligand and receptor both in vitro and in vivo. Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2012R1A2A2A01014898). References [1] P. Anand, A.B. Kunnumakara, C. Sundaram, K.B. Harikumar, S.T. Tharakan, O.S. Lai, B.Y. Sung, B.B. Aggarwal, Cancer is a preventable disease that requires major lifestyle changes, Pharm. Res. 25 (2008) 2097–2116. [2] C.M. Spencer, D. Faulds, Paclitaxel, A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer, Drugs 48 (1994) 794–847. [3] E.K. Rowinsky, N. Onetto, R.M. Canetta, S.G. Arbuck, Taxol: the first of the taxanes, an important new class of antitumor agents, Semin. Oncol. 19 (1992) 646–662. [4] M. Chazard, B. Pellae-Cosset, F. Garet, J.A. Soares, B. Lucidi, Y. Lavail, L. Lenaz, [Taxol (paclitaxel), first molecule of a new class of cytotoxic agents: taxanes], Bull. Cancer 81 (1994) 173–181. [5] M.W. Saville, J. Lietzau, J.M. Pluda, I. Feuerstein, J. Odom, W.H. Wilson, R.W. Humphrey, E. Feigal, S.M. Steinberg, S. Broder, et al., Treatment of HIVassociated Kaposi’s sarcoma with paclitaxel, Lancet 346 (1995) 26–28. [6] B. Ozcelik, C. Turkyilmaz, M.T. Ozgun, I.S. Serin, C. Batukan, S. Ozdamar, A. Ozturk, Prevention of paclitaxel and cisplatin induced ovarian damage in rats by a gonadotropin-releasing hormone agonist, Fertil. Steril. 93 (2010) 1609–1614. [7] H. Gelderblom, J. Verweij, K. Nooter, A. Sparreboom, Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation, Eur. J. Cancer 37 (2001) 1590–1598. [8] J. Gong, M. Chen, Y. Zheng, S. Wang, Y. Wang, Polymeric micelles drug delivery system in oncology, J. Controlled Release 159 (2012) 312–323 (official journal of the Controlled Release Society). [9] F. Danhier, N. Lecouturier, B. Vroman, C. Jerome, J. Marchand-Brynaert, O. Feron, V. Preat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation, J. Controlled Release 133 (2009) 11–17 (official journal of the Controlled Release Society). [10] Y. Lee, R. Graeser, F. Kratz, K.E. Geckeler, Paclitaxel-loaded polymer nanoparticles for the reversal of multidrug resistance in breast cancer cells, Adv. Funct. Mater. 21 (2011) 4211–4218. [11] S. Koudelka, J. Turanek, Liposomal paclitaxel formulations, J. Controlled Release 163 (2012) 322–334 (official journal of the Controlled Release Society).
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Evaluation of polyethylene glycol-conjugated novel polymeric anti-tumor drug for cancer therapy Joung-Pyo Nam 1 , Jun-Kyu Park 1 , Dong-Hee Son, Tae-Hun Kim, Sun-Jeong Park, Seong-Cheol Park, Changyong Choi, Mi-Kyeong Jang, Jae-Woon Nah ∗ Department of Polymer Science and Engineering, Sunchon National University, 255 Juang-ro, Suncheon 540-950, Jeollanam-do, Republic of Korea
a r t i c l e
i n f o
Article history: Received 5 January 2014 Received in revised form 3 April 2014 Accepted 19 April 2014 Available online 22 May 2014 Keywords: Paclitaxel PEG Transferrin Polymeric prodrugs
a b s t r a c t A novel polymeric prodrug (PXPEG) was prepared to enhance the solubility of an anti-cancer drug, paclitaxel, in aqueous solutions and decrease the cytotoxicity by PEGylation, which means PEG attached to another molecule. In addition, the targeting ligand, transferrin (TF), was modified to PXPEG to enhance the therapeutic efficacy. The targeting ligand-modified PXPEG (TFPXPEG) was examined by 1 H-NMR to confirm the successful synthesis. The synthesized TFPXPEG had better solubility than the free drug against aqueous solution. The particle size of TFPXPEG was approximately 197.2 nm and it had a spherical shape. The MTT assay showed that the anti-tumor efficiency of TFPXPEG was better than that of TF-unmodified PXPEG. In the KB tumor-bearing mouse model, the tumor volume of TFPXPEG treated groups was decreased dramatically by more than 2 fold or 3 fold compared to the PBS or PXPEG treated groups. The in vitro and in vivo evaluation showed that TFPXPEG had better efficacy than that of PXPEG due to the targeting effect of targeting ligands, such as TF. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Cancer is known medically as a malignant neoplasm and is comprised of a broad group of diseases involving uncontrolled cell growth. Cancer has genetic or environmental causes. Anand et al. reported that almost 90–95% of all cancer cases can be attributed to environmental factors, whereas the remaining 5–10% of all cancer cases can be attributed to genetic factors [1]. The number of cancer patients is increasing every year due to the changing environmental or genetic factors. Therefore, the number of deaths is also increasing. Many ways to overcome cancer have been reported. Paclitaxel (PX), a mitotic inhibitor, is one of the drugs most used for cancer therapy. PX is effective in clinical trials against a variety of tumors including breast cancer, Kaposi’s sarcoma, ovarian cancer, lung cancer [2–5]. On the other hand, its use should be limited to clinical trials because of the side effects including unusual bruising, pain/swelling/redness at the injection site, fever, cough, dizziness, shortness of breath, skin rash, and female infertility [2,6,7]. Most of these side effects are associated with the excipient used, such as Cremophor EL (CrmEL) and the polyoxyethylated castor oil. The
∗ Corresponding author. Tel.: +82 61 750 3566; fax: +82 61 750 5423. E-mail address: [email protected] (J.-W. Nah). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.04.013 0927-7765/© 2014 Elsevier B.V. All rights reserved.
commercially commonly used formulation, PX (Taxol® ), consists of CrmEL/ethanol (v/v, 50/50), which results in significant side effects [7,8]. Therefore, PX has two major problems when used clinically. One of the problems is the poor solubility and another problem is the side effects. In recent years, extensive studies have been reported to find a way of overcoming the major problems of PX (e.g. nanoparticles [9,10], liposome [11,12], polymeric micelles [13–16], emulsions [17], and polymeric prodrugs [18,19]). The use of polymeric prodrugs has many benefits, such as controlling the release of a drug, altering the drug biodistribution, altering the cellular uptake properties of a drug, taking advantage of the enhanced permeability and retention (EPR) effect, and prolonging the action of a drug [20]. Various bioavailable polymers have been used to prepare the polymeric prodrugs to overcome the disadvantages of the conventional drug. Polyethylene glycol (PEG) is one of the most widely used polymeric prodrugs system for the development of drugs. The structure of PEG is composed of the repeat elements (CH2 CH2 O). PEG imparts water solubility to hydrophobic drugs through PEGylation. PEGylation means PEG attached to another molecule, such a drug or therapeutic protein, via a covalent bond. The PEG moiety of a PEGylated polymeric prodrug has the following specific properties: high mobility in solution, water solubility, lack of toxicity and low immunogenicity, altered distribution in the body, and long circulation times in the body against the reducing renal clearance [21]. Veronese et al. reported
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that the PEG conjugated doxorubicin (anti-cancer agent) had a markedly prolonged plasma clearance and greater tumor targeting compared to free DOX [20]. Pawar et al. reported that PEG provided stability and enhanced aqueous solubility of the final PEGylated conjugate [22]. In this study, PEG-conjugated PX (PXPEG) was prepared to provide enhanced solubility and stability in aqueous solution. In addition, transferrin (TF), the targeting ligand, was conjugated to the side chain of PXPEG (TFPXPEG) for cancer therapy. TF can be bound to the transferrin receptors (TfR) present on the tumor cell surface, which leads to receptor mediated endocytosis. The surface of tumor cells overexpresses TfR because of the urgent requirement of iron to maintain cellular survival and shows 100 times more TfR than normal cells [23,24]. This targeting delivery system can provide lower systemic toxicity and higher therapeutics efficiency. This paper reports the design, synthesis, and in vitro and in vivo evaluation of TFPXPEG with TF as the targeting ligand to enhance the cellular uptake and anti-cancer activity through receptor-mediated endocytosis between TF and TfR. 2. Materials and methods 2.1. Materials The anti-cancer drug model, as a PX, was purchased from SigmaAldrich Chemical Co. (USA). TF, a targeting ligand (from human, 5% in H2 O, ≥98%, 80 kDa) was obtained from Sigma-Aldrich Chemical Co. (USA). COOH–polyethylene glycol–COOH (COOH–PEG–COOH, Mw : 3000 Da) was acquired from Sigma-Aldrich Chemical Co. (USA). Cross linkers, such as 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and N-(3-dimethylaminopropyl) (DMAP), were supplied by SigmaAldrich Chemical Co. (USA). Dulbecco’s modified eagle medium (DMEM), Roswell park memorial institute (RPMI)-1640, trypsin, ethylenediaminetetraacetic acid (EDTA), and fetal bovine serum (FBS) were obtained from Gibco (BRL, MD, USA). All other chemicals and reagents were of extra reagent grade and used as received. 2.2. Methods 2.2.1. Synthesis of paclitaxel–polyethylene glycol polymeric prodrug (PXPEG) PXPEG was synthesized by a chemical reaction using the following method. A predetermined amount of PX (24 mg, 0.028 mmol) and PEG (168.64 mg, 2 equiv.) were dissolved in 1 mL dichloromethane. EDC (2.1 excess) and DMAP (2.1 excess) were then added to the PX and PEG dissolved solution. The reaction was carried out at room temperature for 24 h. After 24 h, the reaction was quenched with water and PXPEG was precipitated in excess diethyl ether. The powder of PXPEG was obtained by drying at 50 ◦ C. This powder was dissolved in distilled water and then it was dialyzed against distilled water for 3 d by using a molecular weight cut-off (MWCO, 3500 g/mol) dialysis membrane to remove the excess reagent. This solution was filtered through a 1.2 m pore sized syringe filter to remove large aggregates. Finally, the PXPEG polymeric prodrug was obtained as a white powder by lyophilization. 2.2.2. Targeting ligand-modified polymeric prodrug (TFPX–PEG) for targeting drug delivery Five milligram (1% of PXPEG, molar ratio) of TF was dissolved in 5 mL distilled water. EDC (2.1 excess) and NHS (2.1 excess) were then added to the TF dissolved solution at room temperature for 6 h with gentle stirring. 20 mg of PXPEG was dissolved in 5 mL of distilled water. A PXPEG solution and NHS-activated TF solution were mixed and reacted with gentle stirring at room temperature for 8 h.
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After the reaction, the resulting solution was introduced into the dialysis membrane (molecular weight cut off, MWCO 3500 Da) and dialyzed against distilled water to remove the unreacted materials, such EDC and NHS for 24 h. The total volume was adapted to 25 mL, and this solution was used to measure an in vitro and in vivo evaluation. In addition, the powder of TFPXPEG was obtained by lyophilization to examine the physicochemical properties. 2.2.3. Characterization of PXPEG and TFPXPEG polymeric prodrugs The polymeric prodrugs, such as PXPEG and TFPXPEG, were characterized by proton nuclear magnetic resonance spectroscopy (1 H-NMR) to confirm the successful synthesis. The1 H-NMR spectra of PXPEG and TFPXPEG were recorded on a 400 MHz NMR spectrometer (AVANCE 400FT-NMR 400 MHz, Bruker) using DMSO-d6 as the locking solvent. PX, PEG, PXPEG, TF, and TFPXPEG (5 mg of each) were dissolved in 0.5 mL of DMSO, and the products were assessed using an NMR spectrometer. The mean particle size and size distribution of the polymeric prodrugs were determined using an ELS-8000 electrophoretic LS spectrophotometer (NICOMP 380 ZLS zeta potential/particle sizer; Otsuka electronics Inc., Japan) equipped with a He–Ne laser beam at a wavelength of 632.8 nm at 25 ◦ C (scattering angle of 90◦ ). A sample solution (0.5 mg/mL) was used for the particle measurements (HE. 013.016-ALU cell adapter) without filtering. PXPEG and TFPXPEG (each 1 mg) was dissolved in 1 mL of deionized water to observe the morphology. Subsequently, 10 L of PXPEG and TFPXPEG suspended in deionized water were placed on a carbon film coated on a copper grid. The grid was stained with 1% uranyl acetate for 30 s and dried for 10 min. The polymeric prodrugs morphology was examined by transmission electron microscopy (TEM, JEOL JEM-2000 FX-II). The PX amounts of the PXPEG and TFPXPEG polymeric prodrugs were determined by high performance liquid chromatography (HPLC) [25]. The HPLC system consisted of a mobile phase delivery pump (LC-20AD HPLC pump, Shimadzu, Japan) and a UV detector (SPD-20A, UV/Vis detector, Shimadzu, Japan). A ZORBAX 300SB-C18 reverse-phase column (250 mm × 4.6 mm, 5 m, Agilent Technologies Inc., USA) was used to separate the components. The mobile phase was composed of acetonitrile and water at a ratio of 40:60 (v/v). The flow rate and column temperature were set to 1.0 mL/min and 30 ◦ C, respectively. The UV absorbance was determined to be 227 nm with an injection volume of 20 L. The amount of PX in PXPEG or TFPXPEG was calculated from standard curves. The assay was linear over the tested concentration range. The drug amount (DA) and grafting efficiency (GE) were calculated as follows: DA% =
weight of the drug in polymeric prodrug × 100% weight of the feeding polymer and drug
GE% =
weight of the drug in polymeric prodrug × 100% weight of the feeding drug
The solubility test of PXPEG and TFPXPEG against deionized water was examined using a UV spectrophotometer (VU1601, Shimazu, Japan). The concentrations of PX, PXPEG, and TFPXPEG were 1 mg/mL based on the amount of PX. 2.2.4. Cells and cell culture conditions The cell lines including human embryonic kidney 293 cells (HEK 293 cell) and cervical adenocarcinoma cells (KB cell) were purchased from KCLB® (Seoul, Republic of Korea). The cells were cultured in RPMI-1640 or DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin in a humidified atmosphere containing 5% CO2 at 37 ◦ C. The cells grown as a monolayer were harvested by trypsinization (trypsin–EDTA).
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2.2.5. In vitro cytotoxicity and anticancer activity assay of polymeric prodrugs A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay of PX, PXPEG, and TFPXPEG was performed to confirm the in vitro cell cytotoxicity and anticancer activity. The experiment was carried out as follows: HEK293 and KB cells were seeded into a 96-well micro plate at a density of 5 × 104 cells/well with 100 L. When the cell confluence reached 80%, the cells were treated with PX, PXPEG, and TFPXPEG for 48 h at equivalent concentrations ranging from 0.001 to 10 g/mL, which were based on the PX amount. After incubation, 10 L of the MTT solution (5 mg/mL in PBS) was added to each well, and the plate was incubated for an additional 2 h at 37 ◦ C. At a determined time, unreacted MTT was removed by aspiration. After removing the MTT-containing medium, the formazan crystals formed in the live cells were dissolved with 100 L DMSO. Finally, the absorbance was measured at 570 nm (optical density) and 670 nm (background subtraction) by VersaMax ELISA Microplate Reader StakMax® . The relative cell
viability (%) was calculated using the following equation [13]; OD value = (OD560 − OD670 )
Relative cell viability (%) =
ODsample − ODblank
(ODcontrol − ODblank )
× 100%
In addition, a TF competition assay was performed in KB cells to confirm the effect of the targeting ligand. The KB cells TFPXPEG were treated with a one-fold excess free TF and the absorbance was measured. 2.2.6. In vivo anticancer activity assay and H&E staining of polymeric prodrugs The xenografted tumors model of TfR overexpressed KB cells were established subcutaneously by injecting 1 × 107 cells into the abdomen of 6- to 8-week-old female athymic nude mice (Orientbio Inc.) to assess the antitumor effect of PXPEG or TFPXPEG. Once the size of the tumors reached 80–100 mm3 , the mice were divided randomly into three groups and injected intravenously with 100 L
Fig. 1. Scheme of novel polymeric prodrugs. (A) PEG conjugated to paclitaxel (PXPEG), (B) targeting ligand as a transferrin (TF) modified to PXPEG (TFPXPEG).
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PBS or PXPEG, or TFPXPEG three times at two day intervals (n = 5, 6.6 mg/kg). The anti-tumor efficacy was analyzed by measuring the size of the tumor using calipers three times weekly until the end of the study. The length and width of the tumor was measured, and the tumor volume was calculated using the following formula: tumor volume = 0.523Lw2 . For histological analysis, the tumor tissue was fixed in 4% formalin, dehydrated in gradual ethanol (50–100%), cleared in xylene, embedded in paraffin, and cut into 5-m sections using a rotary microtome (Leica RM2125 RTS, © Leica biosystems, Germany). Representative sections were stained with hematoxylin (nucleus) and eosin (cytosol), and examined by optical microscopy. For immunohistochemistry, slides were deparaffinized in xylene and processed, as previously described [26]. The histological analysis was visualized by fluorescence microscopy.
stability in the body after a systemic injection. PEG was introduced to PX to obtain the PEG-grafted PX polymeric prodrug (PXPEG). PEG is the most widely used nonionic polymer because of its high aqueous solubility in the field of polymer-based drug delivery [21,27]. Di-carboxyl PEG (Mw : 3000 Da) was used to induce the hydrophobic drug and targeting ligand. PXPEG was synthesized by a coupling reaction between the carboxyl group of PEG and the hydroxyl group of PX. In addition, the targeting ligands, such as a TF, were introduced to PXPEG (TFPXPEG) via a coupling reaction. As shown in Fig. 1, the carboxymethyl group of PEG or PXPEG was activated by EDC and DMAP, which are known as a zero-crossing-linker because the amide bond was formed without leaving a spacer molecular [28]. The PX or TF was reacted with activated-PEG or activatedPXPEG to form the polymeric prodrug or target the polymeric prodrug.
2.2.7. Statistical analysis All data is reported as the mean ± standard deviation (SD) or standard error (SE) unless specified otherwise. The statistically significant differences were determined using Stat View software (Abacus Concepts, Inc., Berkeley, CA), and a Mann–Whitney test (non-parametric rank sum test). A P-value <0.05 was considered significant for evaluating the differences between the treatment groups (* P < 0.05; ** P < 0.01; *** P < 0.001).
3.2. Characterization of novel polymeric prodrugs
3. Result and discussion 3.1. Synthesis of novel polymeric prodrugs A novel polymeric prodrug was synthesized to overcome the low solubility of PX against deionized water and to enhance its
Fig. 2.
1
The formation of polymeric drugs, such as PXPEG and TFPXPEG was confirmed by 1 H-NMR spectroscopy using DMSO DMSO-d6 as the locking solvent. Figs. 2 and 3 show the proton peaks of PX, PEG, PXPEG, TF, and TFPXPEG. As shown in Fig. 2, the peak assignments of PX, PEG, and polymeric drug (PX conjugated to PEG) were classified as follows: PX (Fig. 1A), the proton peaks of CH3 were between 1.0 and 2.3 ppm, the proton peaks CH and CH2 were between 3.6 and 6.3 ppm, and the proton peaks CH of the aromatic group were between 7.2 and 9.0 ppm; COOH–PEG–COOH (Fig. 1B), the proton peaks of CH2 were observed at 3.5 ppm; PXPEG (Fig. 1C), the proton peaks of PX and PEG were confirmed and were chemically shifted due to the steric effect. The shifted peak assignments are as follows: CH2 peak (non-repeat molecular) of PEG, 2.6 and 3.2 ppm
H-NMR spectra of paclitaxel (A), COOH–PEG–COOH (B), and PXPEG (C).
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Fig. 3.
1
H-NMR spectra of PXPEG (A), TF (B), and TFPXPEG (C).
from 3.5 ppm; CH peak of PX (in aromatic group), and 6.8–9.3 ppm from 7.2 to 9.0 ppm. In addition, the peak assignments of TP and TP-modified PXPEG were as follows: TP (Fig. 3B), 0.8–1.3 ppm; TFPXPEG (Fig. 3C), the proton peaks of TF and PXPEG were chemically shifted due to the steric effect. CH2 (non-repeat molecular of PEG) was shifted from 2.6 and 3.2 ppm to 3.5 ppm because the macromolecule as a TF was introduced to the carboxyl group of PEG in PXPEG. 1 H-NMR showed that PXPEG and TFPXPEG were synthesized successfully. The synthesized polymeric prodrugs, such as PXPEG and TFPXPEG, have amphiphilic characteristics due to the hydrophilic portion as a PEG and hydrophobic portion as a PX. These amphiphilic characteristics can form particles such as a micelle by self-aggregation. The micelles were formed by self-aggregation and consisted of a hydrophobic core and hydrophilic shell in an aqueous environment. Therefore, many studies using polymeric micelles for tumor therapeutics have been reported [16,29–31]. As shown in Fig. 4, the particle size distribution (Fig. 4A) and morphology (Fig. 4B) of PXPEG or TFPXPEG were indicated. The particle size distribution and morphology were measured by dynamic light scattering (DLS) and TEM. PXPEG and TFPXPEG showed a unimodal
and narrow particle size distribution (Fig. 4A (a and b)). PXPEG and TFPXPEG had a spherical shape. Table 1 lists the particle size, feed composition, drug amount (DA), grafting efficiency, and the yield of the polymeric prodrugs. When TF was conjugated to PXPEG, the particle size was increased from 127.2 ± 87.3 to 197.2 ± 56.3. The increasing particle size of the polymeric prodrugs was observed by both DLS and TEM. In addition, TF was conjugated successfully to PXPEG and was present on the surface of PXPEG but not inside. The presence of a targeting ligand on the drug carrier can enhance the cellular uptake by receptor-mediated endocytosis [13,22,32]. The DAs of PXPEG and TFPXPEG calculated by HPLC were 11.8% and 10.6%. In particular, the GEs of PXPEG and TFPXPEG were 53.3% and 47.9%, respectively. The yield was less than 50%. The reason for the low yield suggests that the final products were filtered through a 1.2 m pore sized syringe filter to remove the large aggregates. 3.3. Solubility test against aqueous solution Generally, PX has very poor solubility in aqueous solutions; the solubility of PX in water is 0.0003 M. Therefore, the use of PX can cause side effects due to the excipient, such as Cremophor EL
Table 1 Feed composition and characterization of polymeric prodrugs (n = 3). Sample
Amount of PX (mg)
Amount of PEG (mg)
Amount of TF (mg)
DAa (%)
GEb (%)
Yieldc (%)
Particle sized (nm)
PXPEG TFPXPEG
24 24
84.32 84.32
– 5
11.8 10.6
53.3 47.9
41.5 40.3
127.2 ± 87.3 197.2 ± 56.3
a b c d
Degree of drug amount in polymeric prodrugs, calculated by HPLC. Degree of grafting efficiency to PEG, calculated by HPLC. Weight of final product/weight of initial polymer and drug × 100. Determined by DLS measurement.
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Fig. 4. Particle size and morphological observation of polymeric prodrugs. (A) Particle size of PXPEG (a) and TFPXPEG (b) analyzed by DLS. (B) Spherical forms of PXPEG (a) and TFPXPEG (b) were observed by using transmission electron microscope.
(CrmEL) [7,8,14]. In this study, to overcome the poor solubility of PX in aqueous solutions, the polymeric prodrugs were prepared with PEG and TF, which are hydrophilic molecules. This resulted in an increase in the solubility of PX (Fig. 5). The concentration of
each sample (PX, PXPEG, and TFPXPEG) was based on the amount of PX (final PX concentration: 1 mg/mL). The transmittances of PX, PXPEG, and TFPXPEG were 0.61%, 90.10%, and 97.70% at 400 nm, respectively. Interestingly, the hydrophilic molecular-introduced PXPEG and TFPXPEG showed a significant increase in solubility whereas PX was opaque. In addition, TF-conjugated polymeric prodrug (TFPXPEG) showed higher solubility than the other samples, such as PX and PXPEG. This shows that the hydrophilicity of PX was increased due to the introduction of hydrophilic molecules, such as PEG and TF. This shows that the prepared novel polymeric prodrugs can increase the solubility of PX. Forrest et al., reported that the paclitaxel prodrugs prepared by poly(ethylene glycol)-bpoly(-caprolactone) showed high solubility [19]. Lee et al., also reported that hydrophilic molecular (hyaluronic acid (HA) and PEG)-conjugated paclitaxel showed increased solubility [33].
3.4. In vitro cytotoxicity and anti-tumor activity of polymeric prodrugs
Fig. 5. Solubility photography of PX, PXPEG, and TFPXPEG against aqueous solution and transmittance was measured using UV.
A MTT assay was used to measure the cytotoxicity and antitumor activity of the polymeric prodrugs. The normal cells and cancer cells were treated with various formulations of PX, PXPEG, and TFPXPEG polymeric prodrugs at concentrations ranging from 0.001 to 10 g/mL, which are based on the PX amount. As shown in Fig. 6A, the cytotoxicity was examined on the normal cell as HEK293 cells, the cytotoxicity of the PEG and PX-introduced polymeric prodrugs (PXPEG and TFPXPEG) was reduced significantly compared to free PX. The viability of the cells treated with PXPEG and TFPXPEG was more than 60% at all concentrations, whereas the cell viability of PX was less than 60%. In particular, the cell viability of PXPEG and
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Fig. 6. (A) Cytotoxicity of polymeric prodrugs against HEK 293 cells and (B) anti-tumor efficiency of polymeric prodrugs against KB cells in vitro by MTT assay. Each data point represents the mean ± standard deviation (n = 3).
TFPXPEG was 62.2% and 72.8% at high concentrations (10 g/mL), compared to PX (21.2%). In addition, TFPXPEG showed the highest cell viability. The MTT assay showed that PEG and TF decreased the cytotoxicity of PX. As shown in Fig. 6B, the anti-tumor activity of the polymeric prodrugs on the cancer cell line, KB cell, was examined. The cell viability of PXPEG and TFPXPEG decreased with increasing PX concentration, and there were no significant differences between the two drugs. At the lowest concentration (0.001 g/mL), PXPEG and TFPXPEG exhibited efficient anti-tumor effects. In particular, TFPXPEG showed the highest anti-tumor efficiency at all concentrations. This suggests that the TFPXPEG uptake by the cells is higher than PXPEG due to receptor-mediated endocytosis. A previous study reported that TF modified micelles effectively targeted the TfR present on the surface of the cancer cells [13]. Bellocq et al. reported that TF-modified cyclodextrin polymer showed increased transfection efficiency due to the tumor-targeting ability of TF [34]. Fig. 6B shows the results of the competition assay. The TFPXPEG-treated cells after incubation with free TF showed lowest anti-tumor efficiency. The decreased anti-tumor efficiency of TFPXPEG (incubated with free TF) can be explained because of the lower number of binding sites (TfR) that can bind to TF. Zhang et al. also reported that the binding of human TF in Caco-2 cells was apparently inhibited by the addition of a one-fold excess of free TF [35]. The lowest anti-tumor efficiency of TFPXPEG (incubated with free TF) clearly showed TFPXPEG uptake into the cells by endocytosis between TF and TfR. Interestingly, despite the lower number of binding sites on the tumor cells surface, when the dose was increased, TFPXPEG (incubated with free TF) showed similar anti-tumor efficiency to PXPEG. The reason for this is that TFPXPEG has two types of targeted drug delivery: active targeted drug delivery, such as receptor-mediated endocytosis; and passive targeted drug delivery, such as the EPReffect.
PBS, PXPEG, and TFPXPEG, respectively. Similar to the MTT assay, the tumor volume of the PXPEG or TFPXPEG treated group was compared with the PBS treated group. In addition, TF-modified TFPXPEG showed higher effective therapeutic efficacy, more than 2 fold or 3 fold, compared to PXPEG (P < 0.05) or PBS (P < 0.05). This shows that targeting the ligand-modified TFPXPEG is more effective than PXPEG in terms of tumor inhibition due to the active targeting effect. In addition, the histological observations of PXPEG and TFPXPEG polymeric prodrug-treated tumor tissues were carried out after H&E staining to confirm the therapeutic efficacy of the polymeric prodrugs in the KB tumor-bearing mice (Fig. 7B). These histological
3.5. In vivo anti-tumor effect and immunohistological analysis of polymeric prodrugs in KB tumor-bearing mice To evaluate the anti-tumor effects of the PXPEG and TFPXPEG polymeric drugs, the tumor growth rate was examined after an intravascular injection (i.v. injection) of polymeric drugs into the KB tumor-bearing mice (Fig. 7A). The tumor was induced by a subcutaneous injection of KB cells, and PBS, PXPEG and TFPXPEG were injected once the size of the tumors reached 80–100 mm3 in volume. After twenty days from the first treatment day, the mean tumor volume was 2468.3 mm3 , 1742.3 mm3 , and 792.3 mm3 for
Fig. 7. In vivo anti-tumor efficiency and histological analysis of polymeric prodrugs in KB tumor subcutaneous tumor models (n = 5). (A) Inhibition of tumor growth by intravenous injection of PXPEG or TFPXPEG polymeric prodrugs (6.6 mg/kg). * P < 0.05 vs PBS treated group; * P < 0.05 vs PXPEG treated group. (B) Representative photographs of hematoxylin and eosin (H&E) staining in tumor tissues. Original magnification: ×400.
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differences were quantified by the morphometric determination of the proportion of the total tumor area that was necrotic in the H&E staining section [36]. As shown in Fig. 7B, the cells of the PBS-treated group were healthy with a high density. In contrast, the cells in the PXPEG or TFPXPEG polymeric prodrug-treated group showed induced necrosis, and the necrotic area of the tumors treated with the PXPEG or TFPXPEG polymeric prodrugs increased dramatically. In particular, the TFPXPEG treated group showed the largest increase in necrotic area. The MTT assay, tumor volume, and histological observation clearly showed that TFPXPEG can inhibit tumor growth. 4. Conclusion Novel polymeric prodrugs (TFPXPEG) with targeting ligand were prepared. TFPXPEG showed high solubility in an aqueous solution and formed nanoparticles at approximately 197.2 nm. The cytotoxicity of drug decreased when modified by PEG and TF to PXPEG. In addition, the anti-tumor efficiency of the polymeric prodrugs was effective against KB cells in vitro and in vivo. The in vivo and in vitro evaluation clearly showed that the targeting ligand, TF, which was modified to PXPEG, showed therapeutic efficacy and tumor growth inhibition through receptor-mediated endocytosis between TF of TFPXPEG and TfR present on the KB cell surface. Therefore, this study provides a novel polymeric drug for extending the utilization of the PEG-based drug delivery system as well as possible high tumor targeting between the targeting ligand and receptor both in vitro and in vivo. Acknowledgment This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF2012R1A2A2A01014898). References [1] P. Anand, A.B. Kunnumakara, C. Sundaram, K.B. Harikumar, S.T. Tharakan, O.S. Lai, B.Y. Sung, B.B. Aggarwal, Cancer is a preventable disease that requires major lifestyle changes, Pharm. Res. 25 (2008) 2097–2116. [2] C.M. Spencer, D. Faulds, Paclitaxel, A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of cancer, Drugs 48 (1994) 794–847. [3] E.K. Rowinsky, N. Onetto, R.M. Canetta, S.G. Arbuck, Taxol: the first of the taxanes, an important new class of antitumor agents, Semin. Oncol. 19 (1992) 646–662. [4] M. Chazard, B. Pellae-Cosset, F. Garet, J.A. Soares, B. Lucidi, Y. Lavail, L. Lenaz, [Taxol (paclitaxel), first molecule of a new class of cytotoxic agents: taxanes], Bull. Cancer 81 (1994) 173–181. [5] M.W. Saville, J. Lietzau, J.M. Pluda, I. Feuerstein, J. Odom, W.H. Wilson, R.W. Humphrey, E. Feigal, S.M. Steinberg, S. Broder, et al., Treatment of HIVassociated Kaposi’s sarcoma with paclitaxel, Lancet 346 (1995) 26–28. [6] B. Ozcelik, C. Turkyilmaz, M.T. Ozgun, I.S. Serin, C. Batukan, S. Ozdamar, A. Ozturk, Prevention of paclitaxel and cisplatin induced ovarian damage in rats by a gonadotropin-releasing hormone agonist, Fertil. Steril. 93 (2010) 1609–1614. [7] H. Gelderblom, J. Verweij, K. Nooter, A. Sparreboom, Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation, Eur. J. Cancer 37 (2001) 1590–1598. [8] J. Gong, M. Chen, Y. Zheng, S. Wang, Y. Wang, Polymeric micelles drug delivery system in oncology, J. Controlled Release 159 (2012) 312–323 (official journal of the Controlled Release Society). [9] F. Danhier, N. Lecouturier, B. Vroman, C. Jerome, J. Marchand-Brynaert, O. Feron, V. Preat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: in vitro and in vivo evaluation, J. Controlled Release 133 (2009) 11–17 (official journal of the Controlled Release Society). [10] Y. Lee, R. Graeser, F. Kratz, K.E. Geckeler, Paclitaxel-loaded polymer nanoparticles for the reversal of multidrug resistance in breast cancer cells, Adv. Funct. Mater. 21 (2011) 4211–4218. [11] S. Koudelka, J. Turanek, Liposomal paclitaxel formulations, J. Controlled Release 163 (2012) 322–334 (official journal of the Controlled Release Society).
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