Enhanced toxicity and cellular uptake of methotrexate-conjugated nanoparticles in folate receptor-positive cancer cells by decorating with folic acid-conjugated d -α-tocopheryl polyethylene glycol 1000 succinate

Colloids and Surfaces B: Biointerfaces 136 (2015) 383–393

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Enhanced toxicity and cellular uptake of methotrexate-conjugated nanoparticles in folate receptor-positive cancer cells by decorating with folic acid-conjugated d-␣-tocopheryl polyethylene glycol 1000 succinate Varaporn Buraphacheep Junyaprasert a,b,∗ , Sirithip Dhanahiranpruk a , Jiraphong Suksiriworapong a,b , Kittisak Sripha c , Primchanien Moongkarndi d a

Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajathevee, Bangkok 10400, Thailand Center of Excellence in Innovative Drug Delivery and Nanomedicine, Faculty of Pharmacy, Mahidol University, Rajathevee, Bangkok 10400, Thailand c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Rajathevee, Bangkok 10400, Thailand d Department of Microbiology, Faculty of Pharmacy, Mahidol University, Rajathevee, Bangkok 10400, Thailand b

a r t i c l e

i n f o

Article history: Received 15 June 2015 Received in revised form 11 August 2015 Accepted 7 September 2015 Available online 10 September 2015 Keywords: d-␣-Tocopheryl polyethylene glycol 1000 succinate Folic acid Methotrexate Targeted nanoparticles PEGylated poly(␧-caprolactone) MCF-7 breast cancer cells

a b s t r a c t Folic acid-conjugated d-␣-tocopheryl polyethylene glycol 1000 succinate (TPGS–FOL) decorated methotrexate (MTX)-conjugated nanoparticles were developed for targeted delivery of MTX to folate receptor-expressed tumor cells. The synthesis of TPGS–FOL followed 3-step process. Firstly, the terminal hydroxyl group of TPGS was converted to sulfonyl chloride using mesyl chloride in comparison with nosyl and tosyl chlorides. The highest conversion efficiency and yield were obtained by mesyl chloride due to the formation of higher reactive intermediate in a presence of triethylamine. Secondly, the substitution of sulfonyl group by sodium azide produced considerably high yield with conversion efficiency of over 90%. Lastly, the coupling reaction of azido-substituted TPGS and propargyl folamide by click reaction resulted in 96% conjugation efficiency without polymer degradation. To fabricate the folate receptortargeted nanoparticles, 10 and 20%mol MTX-conjugated PEGylated poly(-caprolactone) nanoparticles were decorated with TPGS–FOL. The size and size distribution of MTX-conjugated nanoparticles relatively increased with %MTX. The MTX release from the nanoparticles was accelerated in acidic medium with an increase of %MTX but retarded in physiological pH medium. The decoration of TPGS–FOL onto the nanoparticles slightly enlarged the size and size distribution of the nanoparticles; however, it did not affect the surface charge. The cytotoxicity and cellular uptake of MCF-7 cells demonstrated that 10% MTX-conjugated nanoparticles and FOL-decorated nanoparticles possessed higher toxicity and uptake efficiency than 20% MTX-conjugated nanoparticles and undecorated nanoparticles, respectively. The results indicated that FOL-10% MTX-conjugated nanoparticles exhibited potential targeted delivery of MTX to folate receptor-expressed cancer cells. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Over the past decades, the use of chemotherapeutic drugs has encountered several limitations such as the undesirable biodistribution, the rapid drug clearance and severe adverse effects to normal cells, etc. [1–3]. The active targeting nanoparticles can enhance the drug availability at the target site and reduce the con-

∗ Corresponding author at: Department of Pharmacy, Faculty of Pharmacy, Mahidol University, Rajathevee, Bangkok 10400, Thailand. Fax: +662 644 8694. E-mail address: [email protected] (V.B. Junyaprasert). http://dx.doi.org/10.1016/j.colsurfb.2015.09.013 0927-7765/© 2015 Elsevier B.V. All rights reserved.

centration of intact drug at the healthy site leading to the increased therapeutic efficacy and minimized side effects [4]. To increase efficiency of drug delivery, the nanoparticles should be conjugated with targeting molecules on the surface which can be recognized by the receptors presenting on the cancer cells [5]. One of the most widely used targeting ligands for anticancer drugs is folic acid (FOL) due to its specificity and high binding affinity to the cancer cells. In addition, numerous cancer cells overexpress folate receptors on their cell surface, therefore, the presence of FOL molecules on the surface of nanoparticles can efficiently enhance the cellular uptake via receptor-mediated endocytosis [5–8]. Moreover, FOL provides additional advantages including ease of modification, good stabil-

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ity upon storage and low cost [6]. Therefore, the use of FOL as a targeting agent for anticancer drug delivery to tumors has gained much attention [9]. d-␣-Tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E and polyethylene glycol (PEG) bearing hydrophobic and hydrophilic segments in its structure, respectively [10]. It has been approved by United States Food and Drug Administration (U.S. FDA) to utilize as a drug delivery vehicle. Many reports demonstrated that this polymer could improve the solubility and bioavailability of poorly water-soluble and poorly absorbed drugs by acting as carriers [11–13]. Consequently, the therapeutic efficacy of drug could be enhanced while the side effects of anticancer drugs could be minimized [14,15]. Additionally, TPGS showed inhibitory activity to P-glycoprotein and potent antitumor activity [6,16]. As a consequence, the concomitant administration of TPGS and anticancer drugs, such as doxorubicin, vinblastine and paclitaxel, provided the synergistic effect on cancer cells [17–19]. Nevertheless, a few studies have reported the development of the targeted nanoparticles using FOL-conjugated TPGS (TPGS–FOL) as a targeting moiety [5,20]. The combination of FOL and TPGS in the nanocarriers may synergistically promote the efficiency of targeted nanoparticles to the cancer cells. Methotrexate (MTX) is an extensively used anticancer drug for the treatment of certain human cancers [2,21]. However, the treatment of cancer by MTX requires high-dose regimen and often leads to drug resistance and severe adverse effects [1,2,22]. The minimal toxic side effects and the specificity of MTX to tumor tissues are essential to improve drug safety profile, therapeutic efficacy and patient’s compliance [3]. Although the fabrication of MTX-loaded nanoparticles had been reported, the physical entrapment of MTX in the nanoparticles led to the high amount of MTX burst release within a few hours [23]. Furthermore, the plasma half-life of free MTX is relatively short resulting in the requirement of frequent drug administration [24]. One approach to overcome these problems is to conjugate MTX with macromolecular carriers such as antibodies, polysaccharides, polypeptides and synthetic polymers [25]. For drug-loaded nanoparticles, the covalent binding of drugs along the macromolecules and targeting ligands on the surface of nanoparticles is more desirable because it forms a stable linkage

in the blood circulation [26]. It can prevent the drug leakage prior to the target site and avoid the cleavage of targeting ligand before interacting with the receptor of the target cells. One of interesting chemical reactions is “click reaction” which offers many advantages over other chemical reactions. It is highly stereospecific and simple to conduct under various conditions. In addition, this reaction efficiently and cleanly produces the desired product which may not require purification step [27]. Many researchers have used click reaction for cross-linking of drug/ligand to polymer or polymer to polymer [28,29]. Previously, we reported that the conjugation of MTX along poly(-caprolactone)-co-methoxy poly(ethylene glycol) (P(MTXCLCL)-mPEG) by click reaction has been successfully fabricated without the polymer degradation [30]. However, the synthesis of TPGS–FOL via click reaction has not been established. Furthermore, to the best of our knowledge, the use of MTX-conjugated nanoparticles decorated with TPGS–FOL for the targeted delivery of MTX to the cancer cells has not been reported. The combination of FOL and TPGS may enhance the efficiency of nanoparticles and the conjugation of MTX to the nanoparticles may prevent the drug leakage along the blood stream. Therefore, this study aimed to investigate the conjugation of FOL at the end of TPGS chain by click reaction and to develop the TPGS–FOL decorated MTX-conjugated nanoparticles as targeted nanocarriers in comparison with the undecorated nanoparticles. The uptake efficiency of these nanoparticles to the cancer cells was also investigated in folate receptor-expressed MCF-7 cells. 2. Materials and methods 2.1. Materials TPGS was kindly donated by BASF, Germany. 4-Toluenesulfonyl chloride (tosyl (Ts) chloride, Sigma–Aldrich, Germany), 4nitrobenzenesulfonyl chloride (nosyl (Ns) chloride, Kanto chemical Co., Inc.) and methanesulfonyl chloride (mesyl (Ms) chloride, Kanto chemical Co., Inc.) were used as received. 10% and 20% MTX-conjugated copolymers (10% and 20% P(MTXCLCL)-mPEG) were contributed by Issarachot et al. [30]. FOL, propargylamine, N-hydroxysuccinimide (NHS), copper(II) sulfate, 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide

Scheme 1. Synthetic reaction of TPGS–FOL.

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(MTT), propidium iodide (PI) and trypan blue were purchased from Sigma–Aldrich, Germany. N,N -dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP) and sodium ascorbate were bought from Fluka Chemie, Germany. Dulbecco’s modified eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Biochrom, UK. Triton X-100 was recieved from USB Corporation, USA. Triethylamine (Carlo Erba Reagenti, Italy) was distilled prior to use. Dichloromethane (J.T. Baker, USA) was dried over calcium hydride and distilled before use. Acetonitrile (J.T. Baker, USA) was of high performance liquid chromatography (HPLC) grade. All other reagents and solvents were used as received. 2.2. Synthesis of TPGS–FOL The conjugation of FOL at the terminal chain of TPGS was conducted as described below and schematically illustrated in Scheme 1. 2.2.1. Terminal hydroxyl derivatization of TPGS by sulfonyl derivatives (TPGS–sulfonyl) The terminal hydroxyl group of TPGS was derivatized by aryl or alkyl sulfonyl chloride using Ts chloride, Ns chloride and Ms chloride. Prior to the reaction, TPGS (0.50 g, 0.32 mmol) was dried under vacuum for 2 h and dissolved in 13 mL of dried dichloromethane under nitrogen atmosphere in a reaction flask. Distilled triethylamine (0.25 mL, 1.29 mmol) was added and stirred at room temperature for 30 min. Then, Ts chloride (0.25 g, 1.31 mmol) or Ns chloride (0.29 g, 1.31 mmol) was added while magnetically stirring at room temperature for 12 h. In case of Ms chloride, 0.15 g of Ms chloride (1.31 mmol) was slowly added to the reaction at 0 ◦ C and the reaction was allowed to stir at room temperature for 12 h. After that, the solvent was removed by rotary evaporator. The crude product was re-dissolved in acetone. The mixture was filtered through 0.45 ␮m nylon membrane filter. The saturated sodium bicarbonate solution (1 mL) was added. Finally, the filtrate was dialyzed against deionized (DI) water using a dialysis membrane (MWCO 1000 Da, Spectra Por® , Spectrum laboratories Inc., USA). The dialyzed product was subjected to freeze drying machine (Christ Alpha 1–4, Martin Christ Gefriertrocknungsanlagen GmbH, Germany) yielding a yellowish powder. 2.2.2. Conversion of TPGS–sulfonyl to azido-functionalized TPGS (TPGS–N3 ) TPGS–sulfonyl (0.17 mmol) dissolved in 6 mL of acetonitrile was reacted with sodium azide (0.05 g, 0.77 mmol). The reaction mixture was refluxed at 70 ◦ C under nitrogen atmosphere for 24 h. The resultant product was dialyzed against DI water using dialysis membrane (MWCO 1000 Da) and finally subjected to freeze drying process to attain the dry yellowish powder. 2.2.3. Synthesis of propargyl folamide (PFOL) FOL was functionalized with propargylamine according to the reported method [31] with slight modification. In brief, to the solution of FOL (0.50 g, 1.13 mmol) in 15 mL of anhydrous dimethyl sulfoxide (DMSO), NHS (0.26 g, 2.26 mmol) and DCC (0.28 g, 1.36 mmol) were added under nitrogen atmosphere. The reaction was stirred at 40 ◦ C for 6 h. Subsequently, DMAP (0.16 g 1.36 mmol) and propargylamine (0.08 g, 1.47 mmol) were added and stirred at ambient temperature. After 24 h, the precipitate was removed by filtration. An excess amount of cold diethyl ether was added to obtain yellow precipitates and the remaining diethyl ether was removed under reduced pressure. The final solution was purified through classical column chromatography using siliga gel 60 as a stationary phase and the mixture of absolute ethanol/dichloromethane/28–30 %v/v ammonia solution (60:20:20) as a mobile phase. An eluted solution was dried by

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rotary evaporator. The experiment was conducted in the dark environment. Yield: 52%. 1 H NMR (DMSO-d6): ı (ppm) 8.60 (pteridine), 8.44 (NHCHCOOH), 8.32 (CONHCH2 ), 7.57–6.63 (benzene), 6.93 (NH2 –pteridine), 4.45 (CH2 NH), 4.28 (CONHCHCOOH), 3.80 (CH2 C CH), 3.04 (CH2 C CH), 2.24–1.85 (CH2 CH2 ). 2.2.4. Conjugation of FOL at the terminal chain of TPGS (TPGS–FOL) The azide group of TPGS–N3 was conjugated with PFOL by click reaction using copper(II) sulfate and sodium ascorbate as catalysts. In 2 mL of dried DMSO, TPGS–N3 (0.41 g, 0.26 mmol) was reacted with PFOL (0.25 g, 0.52 mmol). Copper(II) sulfate (9.90 mg, 0.04 mmol) and sodium ascorbate (23.53 mg, 0.12 mmol) were added and stirred at ambient temperature for 24 h under the dark condition. The resultant product was dialyzed against DI water using dialysis membrane (MWCO 1000 Da). The dialyzed product was centrifuged at 6000 rpm for 30 min. The precipitate was washed with DI water and centrifuged again at 6000 rpm for 30 min. The supernatant was discarded and the purified product was then dried under vacuum. 2.3. Characterization The synthesized products were characterized by Fourier transformed infrared (FTIR) spectroscopy, proton nuclear magnetic resonance (1 H NMR) spectroscopy and gel permeation chromatography (GPC). FTIR spectra were obtained from Nicolet 6700 FT/IR spectrophotometer by KBr disc method. 1 H NMR spectra were recorded by Bruker Avance 500 apparatus at 500 MHz, 25 ◦ C in CDCl3 or DMSO-d6 . The GPC chromatograms of the synthesized products were recorded by GPC apparatus (Shimadzu Corporation, Japan) equipped with a refractive index detector using tetrahydrofuran as an eluent and Shodex GPC column as a stationary phase. In case of P(MTXCLCL)-mPEG copolymers, the amount of MTX along the copolymer was quantified after hydrolyzed with a mixture of 50 mM borate buffer pH 9.5 and methanol (1:1 v/v) as reported elsewhere [31]. The unbound MTX in hydrolyzate was analyzed by HPLC method. 2.4. Nanoparticle preparation The MTX-conjugated (P(MTXCLCL)-mPEG) nanoparticles was prepared by dialysis method [31] using 10 and 20 %mol of MTX-conjugated copolymers. To prepare the targeted nanoparticles (FOL-P(MTXCLCL)-mPEG nanoparticles), the MTX-conjugated nanoparticles were decorated with TPGS–FOL. 2.4.1. Preparation of P(MTXCLCL)-mPEG nanoparticles P(MTXCLCL)-mPEG copolymer (7.5 mg) was dissolved in 0.25 mL of dimethylformamide (DMF). Subsequently, this solution was added drop-by-drop into 0.25 mL of DI water under magnetic stirring for 2 h. This mixture was dialyzed against DI water using dialysis membrane (MWCO 1000 Da). During dialysis procedure, the fresh DI water was replaced several times. Then, the resultant dispersion was collected and used for further analysis. 2.4.2. Preparation of FOL-P(MTXCLCL)-mPEG nanoparticles The solution of P(MTXCLCL)-mPEG (7.5 mg) in 0.25 mL of DMF was dropped slowly into 0.35 mL of DI water under magnetic stirring. Subsequently, 0.45 mg of TPGS–FOL in 0.10 mL of DMF was added slowly into the mixture and incubated by magnetically stirring for 2 h. The resultant mixture was dialyzed against DI water using dialysis bag (MWCO 1000 Da). During dialysis procedure, the dialysis medium was changed several times. The colloidal dispersion was collected and used as a dispersion form in the next experiments.

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2.5. Nanoparticle characterization

2.9. In vitro cytotoxicity

The particle size (z-ave), size distribution (PDI) and zeta potential (ZP) of the nanoparticles were measured by Zetasizer NanoZS (Malvern Instrument, UK) at 25 ◦ C. The mean values were obtained by the average of 3 measurements. In case of z-ave and PDI, the measurement was performed at an angle of 173◦ using He–Ne laser at a wavelength of 633 nm. The shape and surface of the nanoparticles were investigated by scanning electron microscopy (Hitachi SEM S-2500, Hitachi, Japan) at an accelerating voltage of 15 kVolt. The nanoparticles were fixed on the stub and coated with gold using sputter–coater prior to the measurement. The processing yield was expressed as %yield and determined after passing the nanoparticles through lyophilization. The %yield was calculated according to Eq. (1).

The viability of MCF-7 cells after incubating with the nanoparticles was evaluated by MTT assay. The MCF-7 cells were seeded at a density of 2 × 104 cells/well in 96-well plate and incubated for 24 h at 37 ◦ C in humidified environment with 5% CO2 . Subsequently, the medium was discarded and the cells were incubated with various concentrations of the nanoparticles (0.0003, 0.003, 0.015, 0.03, 0.15, 0.3, 0.6 and 1.2 mg/mL) for 48 h. Next, the nanoparticles were removed and the cells were washed 3 times with PBS. MTT solution (50 ␮L) was added and incubated at 37 ◦ C for 2 h. After that, the MTT solution was removed and the formazan crystal was dissolved in 100 ␮L of isopropanol. The absorbance values were measured by microplate reader (Infinite 200 NanoQuant, Tecan Group Ltd., Switzerland) at wavelengths of 570 and 650 nm. The cell viability was calculated by the following equation.

%Yield =

Weight of lyophilized nanoparticles × 100 Weight of all solid components in the formulation (1)

2.6. In vitro release study The release behavior of MTX from the P(MTXCLCL)-mPEG nanoparticles was assessed by the published dialysis method [31] using dialysis bag with MWCO of 6000–8000 Da (Cellu-Sep T2, Bioron GmbH, Germany). The nanoparticles (7.5 mg/mL, 2 mL) were filled in the dialysis bag. Subsequently, the tightly sealed dialysis bag was immersed in 30 mL of 0.01 M phosphate buffer solutions pH 4.5 and 7.4. The release study was performed at 37 ◦ C and a rotation speed of 100 rpm. At designated time points, the release medium (2 mL) was withdrawn and replaced with 2 mL of fresh medium immediately. The amount of MTX in the taken sample was quantified by HPLC method. Each formulation was assayed in triplicate (n = 3). 2.7. HPLC analysis of MTX The amount of MTX was analyzed by the reported HPLC method using Shimadzu HPLC machine (Shimadzu Corporation, Kyoto, Japan) [31]. A reverse phase Hypersil ODS column (5 ␮m particle size, 250 × 4.6 mm, Thermo Fisher Scientific Inc., USA) was used as a stationary phase. Phosphate buffer (0.05 M) pH 6 (90%v/v) mixed with acetonitrile (10%v/v) was a mobile phase. The chromatogram was recorded by UV detector at a wavelength of 304 nm. The standard curve was constructed over the concentration range of 0.05–20 ␮g/mL. The HPLC method was validated and accepted in terms of linearity, accuracy and precision.

Cell viability (%) =

AS,570 − AS,650 × 100 AC,570 − AC,570

(2)

where AS,570 and AS,650 are absorbance values of the cells incubated with the nanoparticles measured at 570 and 650 nm, respectively. AC,570 and AC,650 are absorbance values of the cells incubated with culture medium (control) measured at 570 and 650 nm, respectively. 2.10. Quantitative analysis of cellular uptake of nanoparticles In order to quantitatively study the cellular uptake of nanoparticles by MCF-7 cells, coumarin-6 was used as a fluorescence probe. Prior to the experiment, coumarin-6 was loaded in the nanoparticles according to the same procedure as previously described in the nanoparticle preparation section excepting that the probe was dissolved together with P(MTXCLCL)-mPEG copolymer in DMF. The quantitative uptake study was conducted as follows. The MCF7 cells were cultivated in 96-well plate at a density of 4 × 104 cells/well and 37 ◦ C under 5% CO2 atmosphere. After 24 h of incubation, the medium was replaced with 100 ␮L of coumarin-6 loaded nanoparticles at a concentration of 1 mg/mL. At 30 and 120 min, the nanoparticles were removed and the cells were washed 3 times with 50 ␮L PBS. The cells were lysed by 50 ␮L of 0.5%w/v Triton X-100 in 0.2 N NaOH solution. The fluorescence intensity was measured by microplate reader (SpectraMax® Gemini EM, Molecular Devices, LLC, USA) at an excitation wavelength of 430 nm and an emission wavelength of 485 nm. The uptake efficiency is expressed as %uptake and calculated according to Eq. (3).

%Uptake =

Measured fluorescence intensity after cell lysis × 100 Measured fluorescence intensity of the fed nanoparticles

(3)

2.8. Cell culture The cytotoxicity and uptake efficiency of nanoparticles were carried out in folate receptor-expressed MCF-7 breast cancer cells. The cells were cultured in DMEM medium supplemented with 10% FBS, 1% penicillin-streptomycin solution, 1% sodium pyruvate and 0.1% human insulin. They were cultivated in medium at 37 ◦ C in humidified environment with 5% CO2 . The medium was replenished every a few days. After 80–90% confluence, the cells were washed with phosphate buffered saline (PBS) and collected by trypsinization using 1 mM ethylenediaminetetraacetic acid and 0.25% trypsin. The mRNA of folate receptor of MCF-7 cells had been identified by PCR technique and the result confirmed the expression of folate receptor on MCF-7 cells as reported previously [31].

2.11. Qualitative analysis of cellular uptake of nanoparticles The MCF-7 cells were seeded in 6-well plate at a density of 1.7 × 106 cells/well and incubated for 24 h at 37 ◦ C in humidified 5% CO2 environment. One milliliter of coumarin-6 loaded nanoparticles at a concentration of 1 mg/mL was added into the plate. After incubating with the nanoparticles for 120 min, the cells were washed 3 times with PBS and then fixed with 75%v/v ethanol for 15 min. Subsequently, the nuclei of cells were stained with PI solution for 30 min. The cells were further washed 3 times with PBS and observed under confocal laser scanning microscope (Fluo View 1000, Olympus, Japan).

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2.12. Statistical analysis The results are expressed as the mean ± standard deviation (SD) from at least 3 measurements. Statistical tests were performed by using t-test or one way ANOVA for pair or multiple comparisons, respectively. For all tests, p-value less than 0.05 is considered to be statistically significant. 3. Results and discussion 3.1. Synthesis of TPGS–FOL To achieve targeted delivery of MTX to folate receptor-positive cancer cells, the surface of P(MTXCLCL)-mPEG nanoparticles was essentially functionalized by FOL. Based on our proposed nanoparticle architecture (Fig. 1B), the mPEG of P(MTXCLCL)-mPEG and the PEG of TPGS have the different chain length which may lead to the difficulty in the direct surface conjugation. The shorter PEG chain of TPGS may be shielded by the longer mPEG chain of P(MTXCLCL)mPEG. The mPEG chain may thus interrupt the surface conjugation and result in the undesired amount of FOL on the surface of nanoparticles. The use of TPGS–FOL could controllably decorate the P(MTXCLCL)-mPEG nanoparticles with the desired amount of FOL by fixing the initial feeding ratio of TPGS–FOL and P(MTXCLCL)mPEG polymers. Therefore, TPGS–FOL was synthesized prior to the nanoparticle formation in order to use for the decoration of FOL onto the surface of P(MTXCLCL)-mPEG nanoparticles. 3.1.1. Terminal hydroxyl derivatization of TPGS by sulfonyl derivatives (TPGS–sulfonyl) The terminal hydroxyl group at the end of PEG segment of TPGS was derivatized using three different derivatives of sulfonyl chloride, namely Ts chloride, Ns chloride and Ms chloride, for comparison of the derivatization efficiency. The reaction yielded three derivatives of TPGS–sulfonyl (TPGS–Ts, TPGS–Ns and TPGS–Ms, respectively). According to the FTIR spectra of all derivatives of TPGS–sulfonyl (Fig. 2A), the characteristic peak of sulfonyl group was observed at 1180 cm−1 corresponding to S O stretching. Another characteristic peak of S O stretching of sulfonyl group was overlapped by C H bending peaks of TPGS at 1360 cm−1 . Furthermore, the broad peak of terminal hydroxyl group (O H stretching) at around 3400–3600 cm−1 was diminished. Nevertheless, the dramatic reduction of hydroxyl peak intensity was attained in case of TPGS–Ms while the intensity of hydroxyl peak moderately decreased in cases of TPGS–Ns and TPGS–Ts. The structure of sulfonyl derivatives of TPGS was further confirmed by 1 H NMR spectroscopy. In 1 H NMR spectra (Fig. 2B), the characteristic peak of terminal methylene protons at the end of PEG chain was shifted from 4.25 ppm to 4.15–4.38 ppm due to the derivatization of hydroxyl group by sulfonyl group, respectively. The %conversion could be calculated from 1 H NMR spectrum based on the integrals of terminal methylene protons of PEG segment at 4.25–4.38 ppm and methyl protons of hydrophobic side chain of tocopherol at 0.82–0.86 ppm. As summarized in Table 1, the derivatization of hydroxyl group of TPGS gave different %conversion and %yield depending on the type of sulfonyl chloride derivatives. Ms chloride provided the highest %conversion and %yield followed by Ns chloride and Ts chloride, respectively. This result was attributed to the small structure and less steric effect of methyl group of Ms [32,33]. Although the methyl group of Ms exhibited weakly electron donating property, it had relatively small structure and less steric interference to the electron attacking by lone pair electron of hydroxyl group of TPGS. Moreover, it is theoretically established that the addition of TEA as a base in the reaction can cause the derivatization of hydroxyl group

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Table 1 The efficiency results of each reaction for the synthesis of TPGS–sulfonyl derivatives, azido-functionalized TPGS and TPGS–FOL. Copolymer

%Conversiona

Conversion efficiency (%)

%Yield

TPGS–Ts TPGS–Ns TPGS–Ms TPGS–N3 (TPGS–Ts) TPGS–N3 (TPGS–Ns) TPGS–N3 (TPGS–Ms) TPGS–FOL

46.10 60.85 82.85 42.60 58.05 74.80 70.10

– – – 90.44b 91.31b 90.63b 96.27c

58.41 68.46 80.90 99.32 96.38 97.05 90.57

a

%Conversion = [[I4.25−4.38ppm or I3.38ppm or I7.84ppm /2] × 100]/[I0.82−0.86ppm /12],

b

Conversion efficiency (%) =

c

Conversion efficiency (%) =

I3.38ppm of TPGS−N /I0.82−0.86ppm of TPGS−N 3 3 I4.25−4.38ppm of TPGS−sulf onyl /I0.82−0.86ppm of TPGS−sulf onyl I7.84ppm of TPGS−FOL /I0.82−0.86 ppm of TPGS−FOL I3.38ppm of TPGS−N /I0.82−0.86ppm of TPGS−N 3 3

× 100,

× 100.

by Ms chloride to proceed through the elimination-addition reaction [34]. An alpha-hydrogen atom of Ms chloride was attacked by TEA. Consequently, the hydrogen halide was eliminated and resulted in the formation of sulfene intermediate. This intermediate showed higher electrophilicity and reactivity to nucleophilic addition. Therefore, the presence of TEA in the derivatization reaction of Ms chloride led to higher %conversion and %yield of TPGS–Ms. In cases of Ns and Ts, both derivatives did not have the alphahydrogen atom, therefore the derivatization of hydroxyl group by Ns and Ts was conducted through SN 2 nucleophilic substitution reaction. Ns was substituted by nitro group acting as a strong electron withdrawing group whereas Ts was done by methyl group (a weakly electron donating group). Due to the difference in substitution group, Ns exhibited higher electrophile and better reactivity than Ts. Therefore, the derivatization by Ns at the terminal hydroxyl group of TPGS was better than that by Ts. The GPC chromatograms of all TPGS–sulfonyl derivatives illustrated the unimodal peak (Fig. S1) indicating no degradation of TPGS under this condition. The Ms chloride showed the higher derivatization activity than Ns chloride and Ts chloride, respectively.

3.1.2. Conversion of TPGS–sulfonyl to azido-functionalized TPGS (TPGS–N3 ) The sulfonyl group of all TPGS–sulfonyl derivatives was further substituted by azide group using sodium azide. The %yield of all TPGS–sulfonyl derivatives was higher than 96% regardless of the type of TPGS–sulfonyl derivatives (Table 1). After reacting with sodium azide, the FTIR spectra (Fig. 2A, TPGS–N3 ) exhibited the characteristics of azide peak at 2100 cm−1 . Meanwhile, the characteristic peak of sulfonyl group at 1180 cm−1 completely disappeared. The 1 H NMR spectra (Fig. 2B, TPGS–N3 ) showed the change in chemical shift of terminal methylene protons at the end of PEG chain of TPGS from 4.15–4.38 ppm to 3.38 ppm corresponding to the methylene protons adjacent to azide group. The %conversion and conversion efficiency were calculated based on the integrals of terminal methylene protons of PEG segment before and after conversion at 4.25–4.38 ppm and 3.38 ppm, respectively, and methyl protons of hydrophobic chain of tocopherol at 0.82–0.86 ppm. The conversion efficiency of all TPGS–N3 was higher than 90% and comparable among all TPGS–sulfonyl derivatives. Meanwhile, the %conversion of TPGS–N3 directly depended on the %conversion of TPGS–sulfonyl from different sulfonyl derivatives since the different sulfonyl derivatives gave different %conversion in the hydroxyl derivatization step. The highest value of 74.80% was obtained from TPGS–Ms whereas TPGS–Ts showed the lowest one. The GPC chromatograms showed the unimodal peak (Fig. S1). These results suggested that the different TPGS–sulfonyl did neither affect the efficiency of substitution reaction nor cause the degradation of polymer. Based on all results, it can be concluded that the highest

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Fig. 1. Postulated architectures of P(MTXCLCL)-mPEG nanoparticles (A) and FOL-P(MTXCLCL)-mPEG nanoparticles (B).

TPGS–N3 was obtained by using Ms chloride in the derivatization of the terminal hydroxyl group of TPGS. 3.1.3. Conjugation of FOL at terminal chain of TPGS (TPGS–FOL) The azide group of TPGS–N3 and the alkyne group of PFOL were coupled by click reaction using copper(II) sulfate and sodium ascorbate as catalysts. This reaction yielded TPGS–FOL by 90.57% (Table 1). As monitored by FTIR (Fig. 2A, TPGS–FOL), the characteristic peak of azide group at 2100 cm−1 completely disappeared while the characteristic peaks of FOL and triazole linkage were detected in the spectrum. The C N stretching peaks of triazole ring and pteridine ring were overlapped at 1637 cm−1 . The N H and C O stretching peaks of amide bond of FOL were observed at 3264 and 1687 cm−1 , respectively. The 1 H NMR spectrum (Fig. 2B, TPGS–FOL) revealed a new peak at 7.84 ppm corresponding to the methyne proton of triazole ring. Furthermore, the characteristic peaks of FOL were observed in the spectrum. The proton of amide bond and the methylene protons of PFOL appeared at 8.16 and 3.77 ppm, respectively. In addition, the characteristic peaks of FOL also presented in the spectrum over the region of 2.13–2.37 ppm and 4.27–8.63 ppm. The calculation of %conversion and conversion efficiency was based on the integrals of methyne proton of triazole ring at 7.84 ppm, terminal methylene protons of PEG chain at 3.38 ppm and methyl protons of hydrophobic side chain of TPGS at 0.82–0.86 ppm. As shown in Table 1, the %conversion of TPGS–FOL was 70.10% whereas the conversion efficiency was as high as 96.27%. The GPC chromatogram (Fig. S1) showed a shoulder of peak probably due to the presence of remaining unmodified TPGS. The contamination of TPGS occurred during the purification by dialysis since TPGS and TPGS–FOL could not be separated by the dialysis membrane (MWCO 1000 Da). These results led to the conclusion that the conjugation of FOL at the end chain of TPGS was achieved by click reaction without the degradation of TPGS polymer. 3.2. Nanoparticle formation 3.2.1. P(MTXCLCL)-mPEG nanoparticles According to the analysis of MTX content in MTX-conjugated copolymers, the amounts of MTX in 10% and 20% P(MTXCLCL)mPEG copolymers were 4.5 and 10.6 mg/g of copolymer, respectively. Table 2 summarizes the characteristics of P(MTXCLCL)mPEG nanoparticles. In this study, P(CL)-mPEG nanoparticles were prepared for comparison. From the results, the particle size of both 10% and 20% P(MTXCLCL)-mPEG nanoparticles was significantly larger than that of P(CL)-mPEG nanoparticles due to the presence of drug in the nanoparticle core. Moreover, the particle size

of 20% P(MTXCLCL)-mPEG nanoparticles was significantly larger than 10% P(MTXCLCL)-mPEG nanoparticles owing to an increasing amount of conjugating MTX available in the nanoparticle core [35]. The PDI value of both 10% and 20% P(MTXCLCL)-mPEG nanoparticles was slightly different from that of P(CL)-mPEG nanoparticles. Both 10% and 20% P(MTXCLCL)-mPEG nanoparticles showed negative surface charge. In addition, their ZP values were not different from that of P(CL)-mPEG nanoparticles indicating that the core of the P(CL) segment would be surrounded by a hydrophilic outer shell of the PEG segment (Fig. 1A). This structure supported that the nanoparticles could avoid the interaction with serum proteins and the macrophageal uptake in the circulation, leading to the longevity of nanoparticles in the blood stream [36]. Using the dialysis method, the %yield of all nanoparticles including P(CL)-mPEG nanoparticles was around 50–60% and decreased with increasing %conjugating MTX. This was probably resulted from the aggregation tendency of MTX-conjugated polymer containing higher amount of drug. The %drug loading and %loading efficiency of MTX were 0.45 ± 0.02% and 58.93 ± 2.35% for 10% P(MTXCLCL)mPEG nanoparticles and 1.06 ± 0.06% and 54.40 ± 3.18% for 20% P(MTXCLCL)-mPEG nanoparticles, respectively (Table 2). The SEM micrographs of P(MTXCLCL)-mPEG nanoparticles displayed spherical as seen in Fig. 3A and C. The release characteristics of MTX from 10% and 20% P(MTXCLCL)-mPEG nanoparticles were investigated as shown in Fig. 4. The result revealed that the release pattern of MTX was biphasic with an initial burst release within the first 24 h and the subsequent sustained manner over 20 days. The initial fast release was probably caused by the release of drug molecules being at or nearby the nanoparticle surface. The gradual release over 20 days arose from the slow cleavage of amide linker between drug and polymer and the tardy diffusion of liberated drug through the hydrophobic nanoparticle core [37]. The total extent of MTX released from both nanoparticles in phosphate buffer pH 7.4 was less than 30% and lower than that in medium pH 4.5 as a consequence of undergoing acid-catalyzed hydrolysis of amide linkage [38]. In buffer pH 4.5, the release of drug tended to depend on %conjugating MTX. The pH-dependent release characteristics of these nanoparticles may be efficient in tumor targeting after internalization into the cells. The drug may be released faster with higher extent in acidic environment of endosomal vesicle. Meanwhile, the leakage of drug from the nanoparticles may be prevented in the blood circulation [38]. In addition, the conjugation of MTX on the polymer backbone may overcome the MTX resistance by cancer cells due to an alteration of the uptake of intact MTX [22]. Although the total release of MTX from the nanoparticles was less than 50%,

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389

Fig. 2. FTIR (A) and 1 H NMR (B) spectra of TPGS–sulfonyl derivatives, azido-functionalized TPGS (TPGS–N3 ) and TPGS–FOL. Table 2 Particle size (z-ave), polydispersity index (PDI), zeta potential (ZP), %yield, %MTX loading, %loading efficiency, in vitro cytotoxicity and %uptake of nanoparticles (n ≥ 3). Formulations

z-ave (nm) PDI

ZP (mV)

%Yield

%MTX loadinga

%Loading efficiencya

IC50 equivalent to %Uptake of MTXb (␮g/mL) nanoparticlesb 30 min

P(CL)-mPEG 10% P(MTXCLCL)-mPEG FOL-10% P(MTXCLCL)-mPEG 20% P(MTXCLCL)-mPEG FOL-20% P(MTXCLCL)-mPEG

178 254 275 339 374

± ± ± ± ±

*

8 5* 7** 3* 12**

0.233 0.156 0.227 0.291 0.306

± ± ± ± ±

0.042 –15.9 0.003* −13.9 0.012** −13.9 0.008* −12.5 0.044 −11.7

± ± ± ± ±

0.3 0.2 0.1 0.3 0.3

59.72 58.93 56.67 54.40 52.71

± ± ± ± ±

c

3.83 ND 2.35 0.45 2.97 0.40 3.18 1.06 3.53 0.94

c

± ± ± ±

0.02 0.02 0.06 0.06

ND 58.93 53.46 54.40 49.73

c

± ± ± ±

2.35 2.80 3.18 3.33

The values are expressed as the mean ± SD from at least three experiments. a Calculated based on the analyzed amount of MTX and the actual weight of lyophilized nanoparticles. b Determined on MCF-7 cells, c Not determined, * Significantly different among the undecorated nanoparticles (p-value < 0.05), ** Significantly different comparing with the undecorated nanoparticles at the same %MTX conjugating (p-value < 0.05), *** Statistically significant difference comparing between 30 and 120 min of incubation time (p-value < 0.05).

ND 0.167 0.053 0.287 0.147

120 min

c

± ± ± ±

0.022 0.012 0.027 0.060

ND 14.8 19.9 13.4 19.1

± ± ± ±

5.3 5.9 3.1 8.0

NDc 34.7 51.8 23.3 43.4

± ± ± ±

4.0*** 5.3**,*** 3.6*** 7.8**,***

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Fig. 3. SEM photographs of 10% and 20% P(MTXCLCL)-mPEG nanoparticles (A and C, respectively) and FOL-10% and FOL-20% P(MTXCLCL)-mPEG nanoparticles (B and D, respectively).

the release of drug can be accelerated by high amount of enzymes in the target tumor tissues [39,40]. In addition, the slow release with pH-dependent manner may possibly prolong the systemic circulation, lower the drug elimination in in vivo and increase the extracellular drug concentration at the tumor site [7]. Therefore, the slow release of MTX from 10% and 20% P(MTXCLCL)-mPEG nanoparticles is beneficial in the drug delivery of MTX. 3.2.2. FOL-P(MTXCLCL)-mPEG nanoparticles The FOL-decorated MTX-conjugated nanoparticles (FOL-10% and FOL-20% P(MTXCLCL)-mPEG nanoparticles) were prepared on the purpose of tumor targeting. TPGS–FOL was used to enhance the uptake and cytotoxicity in folate receptor-positive cancer cells. In addition, the use of TPGS–FOL to form the nanoparticles may prevent the dissociation of TPGS–FOL from the nanoparticles. Due to the hydrophobic nature of tocopherol of TPGS, the tocopherol segment of TPGS–FOL could form the strong hydrophobic interaction inside the core of the nanoparticles (Fig. 1B). Meanwhile, the FOL-conjugated PEG chain of TPGS formed the hydrophilic corona together with mPEG segment of P(MTXCLCL)-mPEG. After preparation, the particle size and PDI of both FOL-10% and FOL-20% P(MTXCLCL)-mPEG nanoparticles slightly increased as compared to the corresponding undecorated nanoparticles (Table 2). These results related to the insertion of tocopherol segment into the hydrophobic part of the nanoparticles. The surface charge of FOLdecorated MTX-conjugated nanoparticles was still negative. The ZP values remained unchanged as compared to those of undecorated ones. According to the proposed architecture in Fig. 1B,

the mPEG chain of P(MTXCLCL)-mPEG could shield the charge of deprotonated carboxyl group of TPGS–FOL due to the shorter PEG chain of TPGS. The decoration of P(MTXCLCL)-mPEG nanoparticles by TPGS–FOL did not affect the yield of FOL-P(MTXCLCL)-mPEG nanoaprticles. The %MTX loading and loading efficiency of FOLP(MTXCLCL)-mPEG nanoparticles were comparable with those of P(MTXCLCL)-mPEG nanoparticles. In addition, the FOL contents of FOL-10% and 20% P(MTXCLCL)-mPEG nanoparticles were found to be 0.50 ± 0.03 and 0.47 ± 0.03 %␮mol, respectively. The SEM micrograph showed the morphology of the nanoparticles as shown in Fig. 3B and D. The FOL-decorated P(MTXCLCL)-mPEG nanoparticles were spherical in shape. 3.3. In vitro cytotoxicity of nanoparticles 3.3.1. In vitro cytotoxicity of P(MTXCLCL)-mPEG nanoparticles To confirm the activity of conjugating MTX against tumor cells, the cytotoxicity of P(MTXCLCL)-mPEG nanoparticles on MCF-7 cells was studied by MTT assay. The result from the cytotoxicity study is expressed as an IC50 value which is an equivalent concentration of MTX in nanoparticles diminishing MCF-7 cell viability by 50%. The MTX solution was also evaluated and used as a comparative control. As summarized in Table 2, the P(MTXCLCL)mPEG nanoparticles showed significantly lower IC50 value than the MTX solution (IC50 = 1.682 ± 0.115 ␮g/mL). This result indicated that the nanoparticles could enhance the toxicity of MTX to MCF-7 cells by 10.1 and 5.9 times for 10% and 20% P(MTXCLCL)mPEG nanoparticles, respectively. The 10% P(MTXCLCL)-mPEG

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Fig. 4. Release profiles of MTX from 10% and 20% P(MTXCLCL)-mPEG nanoparticles in phosphate buffer pH 4.5 (A) and 7.4 (B) at 37 ◦ C. An error bar indicates the standard deviation from three measurements.

nanoparticles illustrated significantly lower IC50 value than the 20% P(MTXCLCL)-mPEG nanoparticles indicating higher toxicity of 10% P(MTXCLCL)-mPEG nanoparticles due to their smaller size as compared to 20% P(MTXCLCL)-mPEG nanoparticles. 3.3.2. In vitro cytotoxicity of FOL-P(MTXCLCL)-mPEG nanoparticles The IC50 value of FOL-P(MTXCLCL)-mPEG nanoparticles was also determined to investigate the effect of FOL decorated on P(MTXCLCL)-mPEG nanoparticles on the toxicity to MCF7 cells. The result in Table 2 revealed that the IC50 values of FOL-P(MTXCLCL)-mPEG nanoparticles were lower than those of P(MTXCLCL)-mPEG nanoparticles. This result indicated the enhanced toxicity of FOL-P(MTXCLCL)-mPEG nanoparticles by 1.5 and 1.9 times for FOL-10% and 20% P(MTXCLCL)-mPEG nanoparticles although their particle size was larger than that of undecorated nanoparticles. In addition, the shielding effect of mPEG on the appearance of carboxyl group of FOL on the surface of nanoparticles did not have a significant impact on the toxicity of nanoparticles. Hence, the cytotoxicity of P(MTXCLCL)-mPEG nanoparticles could be potentiated by decorating with TPGS–FOL whose FOL moiety could serve as a targeting ligand. 3.4. Uptake study of P(MTXCLCL)-mPEG and FOL-P(MTXCLCL)-mPEG nanoparticles by MCF-7 cells To better understand the enhanced toxicity and evaluate the targeting efficiency of P(MTXCLCL)-mPEG and FOL-P(MTXCLCL)mPEG nanoparticles, the uptake of these nanoparticles was quantified in MCF-7 cells. In this study, coumarin-6 used as a fluorescence probe was loaded in the nanoparticles. Prior to the

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uptake study, the release of coumarin-6 from the nanoparticles was determined to be less than 3% after incubating in the culture medium for 120 min at 37 ◦ C (Table S1), implying that coumarin-6 attached to the nanoparticles throughout the experiment. In Table 2, the result of cellular uptake is expressed as %uptake referring to the uptake efficiency of nanoparticles by MCF-7 cells. After incubating for 30 min, the uptake efficiency of 10% and 20% P(MTXCLCL)-mPEG nanoparticles was determined to be comparable (p-value > 0.05). Likewise, the decoration by TPGS–FOL did not affect the uptake efficiency of these nanoparticles at 30 min of incubation (p-value > 0.05). Nevertheless after 120 min, the uptake of all nanoparticles significantly increased by at least two times higher than that at 30 min. This result indicated that the uptake of FOL-decorated and undecorated nanoparticles underwent the time-dependent process. The 10% P(MTXCLCL)-mPEG nanoparticles were taken up by MCF-7 cells significantly higher than the 20% P(MTXCLCL)-mPEG nanoparticles due to the smaller size of 10% P(MTXCLCL)-mPEG nanoparticles. In addition, the surface presenting FOL by decorating with TPGS–FOL could significantly improve the uptake of the nanoparticles even though the FOL-P(MTXCLCL)-mPEG nanoparticles had larger particles than the P(MTXCLCL)-mPEG nanoparticles. Regarding the shielding effect of mPEG on the carboxyl group of FOL, the result suggested no significant effect on the uptake of the nanoparticles as well as the interaction between FOL molecule and folate receptor on the cell membrane. These results indicated that the decoration of P(MTXCLCL)-mPEG nanoparticles by TPGS–FOL could efficiently enhance the nanoparticle uptake by folate receptor-expressed MCF-7 cells possibly via receptor mediated endocytosis [31]. Similar to the undecorated nanoparticles, the FOL-10% P(MTXCLCL)-mPEG nanoparticles showed higher uptake efficiency than the FOL-20% P(MTXCLCL)-mPEG nanoparticles. Because the FOL contents of FOL-10% and 20% P(MTXCLCL)-mPEG nanoparticles were comparable, higher uptake of FOL-10% MTXconjugated nanoparticles was due to their smaller size which could facilitate the internalization by the cells [41,42]. The quantitative cellular uptake results at 120 min of incubation were consistent with the in vitro cytotoxicity data at 48 h. The cellular uptake at 120 min and in vitro cytotoxity of 10% P(MTXCLCL)-mPEG nanoparticles were higher than those of 20% P(MTXCLCL)-mPEG nanoparticles. Furthermore, both FOLdecorated 10% and 20% P(MTXCLCL)-mPEG nanoparticles showed greater efficiency than the undecorated nanoparticles. From these results, it could be summarized that FOL-10% P(MTXCLCL)-mPEG nanoparticles obviously improved the cellular uptake and cytotoxicity to MCF-7 cells due to FOL moiety serving as a targeting ligand. To further confirm that the nanoparticles were internalized by MCF-7 cells, the cellular uptake of nanoparticles was qualitatively visualized by confocal laser scanning microscope as illustrated in Fig. 5. It was found that the intense green fluorescence of courmarin-6 surrounded the nucleus having red fluorescence. The fluorescence intensity of 10% P(MTXCLCL)-mPEG nanoparticles was stronger than that of 20% P(MTXCLCL)-mPEG nanoparticles (Fig. 5 Rows 1 and 3, respectively). Additionally, the FOL-decorated nanoparticles yielded stronger fluorescence intensity than the undecorated nanoparticles (Rows 1 versus 2 and Rows 3 versus 4, respectively). This finding agreed well with the quantitative uptake results. From the cellular uptake and cytotoxicity results, it evidently indicated that FOL-decorated P(MTXCLCL)-mPEG nanoparticles provided the targeting efficiency on MCF-7 cells for the MTX delivery and 10% MTX-conjugated nanoparticles illustrated higher significant effect on MCF-7 cells than 20% MTX-conjugated nanoparticles.

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Fig. 5. Confocal laser scanning microscopic images of MCF-7 cancer cells after 120 min of incubation with the coumarin-6 loaded nanoparticles. Rows 1 and 3 are the images of cells incubated with 10% and 20% P(MTXCLCL)-mPEG nanoparticles while rows 2 and 4 are those incubated with FOL-10% and FOL-20% P(MTXCLCL)-mPEG nanoparticles, respectively. Left and middle columns are images of coumarin-6 and PI channels, respectively. Right column illustrates the overlapping images of coumarin-6 and PI channels.

4. Conclusion

Acknowledgements

The present study showed the successful reaction for conjugating TPGS with FOL. Firstly, Ms chloride showed the highest efficiency in TPGS derivatization. The subsequent substitution and the conjugation of PFOL to TPGS–N3 through click reaction were feasible and succeeded without polymer degradation. The MTX-conjugated nanoparticles without and with the decoration of TPGS–FOL were fabricated for the tumor targeted delivery of MTX. The increased %conjugating MTX and the decoration by TPGS–FOL enlarged the particles and widened the size distribution of nanoparticles. However, 10% conjugating MTX possessed more potent cytotoxicity and higher uptake efficiency by MCF-7 cells than 20% MTX-conjugated nanoparticles. Furthermore, the decoration of P(MTXCLCL)-mPEG nanoparticles by TPGS–FOL potentiated cytotoxicity and cellular uptake efficiency. This study demonstrated that FOL-decorated MTX-conjugated nanoparticles are the potential carriers for targeting delivery of MTX to folate receptorexpressed MCF-7 cells.

The authors gratefully acknowledge research grants from Mahidol University and from The Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative for financial support. We thank Dr. Ousanee Issarachot for her helpful contribution of MTX-conjugated polymers. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.09. 013. References [1] A.J. Mazur, D. Nowak, H.G. Mannherz, M. Malicka-Blaszkiewicz, Eur. J. Pharmacol. 613 (1–3) (2009) 24–33.

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