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Vitamin E succinate-grafted-chitosan oligosaccharide/RGD-conjugated TPGS mixed micelles loaded with paclitaxel for U87MG tumor therapy Yanzuo Chen, Shu Feng, Wenchao Liu, Zeting Yuan, Peihao Yin, and Feng Gao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01068 • Publication Date (Web): 17 Feb 2017 Downloaded from http://pubs.acs.org on February 18, 2017

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Molecular Pharmaceutics

Vitamin E succinate-grafted-chitosan oligosaccharide/RGD-conjugated TPGS mixed micelles loaded with paclitaxel for U87MG tumor therapy Yanzuo Chen1, Shu Feng1, Wenchao Liu1, Zeting Yuan3, Peihao Yin3, Feng Gao* 1,2 1

Department of Pharmaceutics, School of Pharmacy, East China University of

Science and Technology, Shanghai 200237, China 2

Shanghai Key Laboratory of Functional Materials Chemistry, School of

Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China 3

Interventional Cancer Institute of Chinese Integrative Medicine, Putuo Hospital,

Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China

* Corresponding author. Department of Pharmaceutics, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China. E-mail address: [email protected] (F. Gao) 1

ACS Paragon Plus Environment


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Abstract The poor therapeutic efficacy of hydrophobic chemotherapeutic drugs is an intrinsic limitation to successful chemotherapy. In the present study, a multi-task delivery system based on arginine-glycine-aspartic acid peptide (RGD) decorated vitamin E succinate (VES)-grafted-chitosan oligosaccharide (CSO)/RGD-conjugated D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS-RGD) mixed micelles (VeC/T-RGD MM) was first prepared for targeted delivery of a hydrophobic anticancer drug-paclitaxel (PTX) to improve the efficacy of U87MG tumor therapy. VES grafted CSO (VES-g-CSO) and TPGS-RGD were synthesized as nanocarriers, and PTX loaded VeC/T-RGD MM (PTX@VeC/T-RGD MM) was prepared via the organic solvent emulsification-evaporation method. The PTX@VeC/T-RGD MM was 150.2 nm in diameter with uniform size distribution, 5.92% drug loading coefficient and no obvious particle size changes within 7 days. The PTX@VeC/T-RGD MM showed sustained-release properties in vitro and high cytotoxicity, and could be efficiently taken up by human glioma U87MG cells. The tumor inhibitory rate of PTX@VeC/T-RGD MM treatment in U87MG tumor spheroids and U87MG tumor-bearing mice was 49.3% and 88.4%, respectively, which indicated a superior therapeutic effect. PTX@VeC/T-RGD MM did not damage normal tissues in safety evaluations. These findings suggested that PTX@VeC/T-RGD MM could be developed for the delivery of hydrophobic drugs to U87MG tumors.

Keywords: VES-grafted-CSO, TPGS-RGD, PTX, mixed micelles, U87MG tumor inhibition

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Introduction The mortality of cancer, more than HIV/AIDS, tuberculosis, and malaria combined, is in the lead worldwide.1 Although chemotherapy has shown promising results, the survival of patients is compromised mainly by the poor selectivity of chemotherapeutic drugs, which often cause injury to normal tissues.2 In the last 30 years, several nanotechnology-based drug delivery systems3-5 have been widely investigated6. The micelle system has attracted increasing attention as a nanocarrier for antitumor drug delivery because of its solubilization properties, technical ease, and high biocompatibility.7 However, the common micelle still has many defects, such as low drug loading capacity and poor stability. To overcome these problems, the mixed micelle system was developed.8 Mixed micelles have a more compact structure than common micelles, which provide a better hydrophobic micro-environment and allow encapsulation and stabilization of hydrophobic agents in their inner core for solubilization, thus enhancing the bioavailability of these agents and prolonging circulation time.9-10 The mixed micelles were allowed to passively accumulate at the tumor site according to size 11 via enhanced permeability and retention effects because of the leaky vasculature.12 In addition, mixed micelles enhance the cytotoxicity and increase the blood circulation time of chemotherapeutic agents.13 However, low drug loading coefficients (DL) remain a challenge in most mixed micelle systems.14 To overcome these issues, a strategy based on the selection of proper copolymers containing compatible hydrophobic segments was proposed.15 Chitosan has excellent biocompatibility, biodegradability, and bioactivity, as well as low toxicity.16 However, chitosan is insoluble at pH values above its pKa (pH 6.4), and amphiphilic chitosan micelles would precipitate under physiological condition. Chitosan oligosaccharide (CSO), which has better solubility than chitosan, could serve as the “patch board” and offers many reaction sites for further modification, making it a potential component in the preparation of drug delivery systems. Studies have shown that CSO could be considered non-toxic,17 and associated with the reduction of blood cholesterol and antitumor properties.18 CSO could be decorated by many other chemical groups to prepare CSO micelles for clinical use. In addition, advanced material studies on CSO 3



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micelles have promoted their research, and CSO micelles have shown promising results for clinical application in the treatment of tumors, as well as in pre-clinical development, such as wound healing19 and antitumor effects20-21. VES is a hydrophobic vitamin E analogue and an excellent solvent for hydrophobic drugs. VES could be grafted on the CSO backbone to form an amphiphilic copolymer, and then VES-g-CSO could self-assemble to form micelles for hydrophobic drug delivery. TPGS shares the same hydrophobic segment with VES; it also has some properties that enhance the cellular uptake of drugs.22 Mixed micelles consisting of VES-grafted-CSO (VES-g-CSO) and TPGS are designed to encapsulate hydrophobic drugs. Furthermore, the introduction of TPGS into the drug delivery system could improve drug solubilization23 and reduce the surface charge because of its negative charge, which improves colloidal stability and decreases toxicity to normal cells.1 Studies have described the development of CSO-based mixed micelles. Dou et al. constructed docetaxel-loaded stearic acid-grafted-CSO/TPGS mixed micelles;10 tocopheryl succinate-conjugated CSO encapsulated PTX was designed by Tao.24 However, these CSO mixed micelles do not possess targeting capabilities. The development of tumor targeting delivery systems provides potential to overcome this problem.25 Considering the strong affinity between targeting ligands and specific receptors that are overexpressed on the malignant cells, arginine-glycine-aspartic acid peptides (RGD)-conjugated TPGS (TPGS-RGD) was synthesized as a component of mixed micelles to enhance the anti-tumor efficiency and decrease the toxicity to normal tissues. RGD is an adhesion motif of extracellular matrix proteins in integrin, especially αvβ3.26 The αvβ3 integrin receptors, expressed on U87MG cells, could activate endothelial cells, and make RGD a potential ligand for tumor therapy.27 Paclitaxel (PTX), a classical chemotherapeutic drug, is clinically approved for cancer treatment. However, its clinical application is limited by poor hydrophilicity, unfavorable therapeutic index, and commonly occurring drug resistance.14,20 In the present study, PTX was selected as a model drug for investigation, and a multi-task delivery platform consisting of PTX loaded VES-g-CSO/TPGS-RGD mixed micelles (PTX@VeC/T-RGD MM) was constructed to reduce the surface charge, enhance the 4


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DL of PTX, confer targeting properties to mixed micelles, and improve the U87MG tumor therapeutic effects. The physicochemical characteristics of PTX@VeC/T-RGD MM were investigated. U87MG cellular uptake, apoptosis, and tumor inhibition in vitro, ex vivo, and in vivo were analyzed to determine the evaluation of the efficacy of PTX@VeC/T-RGD MM for the treatment of U87MG tumors.

2.

Materials and methods

2.1 Materials and animals Chitosan oligosaccharide (CSO, MW 5 kDa, 90.0% deacetylation degree) was obtained from Aoxing Co. Ltd. (Zhejiang, China). Vitamin E succinate was purchased from TCI Development Co. Ltd. (Shanghai, China). D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS), succinic anhydride (SA), N-hydroxysuccinimide (NHS), 4-dimethylaminopyridine (DMAP), N,N-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

(EDC),

triethylamine

(TEA),

coumarin-6 were purchased from Sigma (St. Louis, MO, USA). Paclitaxel (PTX) was obtained from Shandong Pansense Pharmaceutical Co. Ltd. (Shandong, China), PTX solution was prepared according to the Taxol®. c(RGDfK) was obtained in GL Biochem Ltd. (Shanghai, China). Purified deionized water was prepared by Milli-Q plus system (Millipore Co., Billerica, MA, USA). All the other solvents used were analytical grade. Fetal bovine serum (FBS), phosphate buffered saline (PBS), 0.25% (w/v) trypsin solution, penicillin-streptomycin and Dulbecco’s modified Eagle’s medium (DMEM) were

purchased

from

Gibco

BRL

(Gaithersberg,

3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium

bromide

MD, (MTT)

USA). was

purchased from Sigma (St. Louis, MO, USA). Annexin V-FITC/PI Apoptosis Detection Kit and 4,6-diamidino-2-phenylindole (DAPI) were provided by Beyotime Biotechnology Co. Ltd. (Nantong, China). Alexa Fluor 594 anti-mouse CD31 antibody was obtained from Biolegend (San Diego, CA, USA). The U87MG cell line was bought from Shanghai Institute of Cell Biology, and was incubated in DMEM medium containing 10% (v/v) FBS and 1% penicillin-streptomycin at 37°C, under 5% 5



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CO2 (90% relative humidity). Before the experiment, cells were pre-cultured until a confluence of 80%-90% was reached. Male BALB/c nude mice (20 ± 2) g, purchased from the Department of Experimental Animals, Fudan University (Shanghai, China), were treated according to the protocols evaluated and approved by the Ethical Committee of the Fudan University. 2.2 Synthesis and characterization of VES-g-CSO and TPGS-RGD 2.2.1 Synthesis of VES-g-CSO VES-g-CSO was synthesized according to previous reports with minor modifications.28 CSO (0.67 g, 4 mmol) was dissolved in 30 mL water at room temperature. VES (0.38 g, 0.72 mmol), EDC (1.38 g, 7.2 mmol) and NHS (0.82 g, 7.2 mmol) were dissolved in 30 mL anhydrous ethanol, and then added into the CSO solution drop by drop, followed by stirring in the dark for 24 h (25°C). The resulting solution was concentrated in a vacuum, and precipitated in cold anhydrous diethyl ether. The product was collected and washed with anhydrous ethanol three times. Finally, the ethanol was evaporated in a vacuum (40°C) overnight to obtain the product. 2.2.2 Synthesis of TPGS-RGD TPGS (1.51 g, 1 mmol), SA (0.40 g, 4 mmol) and DMAP (0.12 g, 1 mmol) were dissolved in 15 mL of methylene dichloride and stirred for 24 h at 25°C. Then the mixture was transferred to -18°C for 4 h and filtered. The resulting filtrate was dialyzed using a dialysis bag (MWCO = 1 kDa, Greenbird Inc., Shanghai, China) against 50% ethanol for 24 h and deionized water for another 48 h to remove the excess SA and DMAP (room temperature). The resulting TPGS-SA was obtained after evaporating the solvent of the dialysate at 50°C, and preserved at 4°C. TPGS-SA (1.63 g, 1 mmol), NHS (0.51 g, 4 mmol) and DCC (0.82 g, 4 mmol) were dissolved in 10 mL of methylene dichloride and stirred for 24 h (25°C) under a nitrogen atmosphere. The mixture was filtered and precipitated by cold diethyl ether. The intermediate product TPGS-NHS was preserved at 4°C. TPGS-NHS was dissolved in 1 mL DMF and stirred for 24 h at 25°C. Then RGD 6


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(0.04 g, 0.06 mmol) was dissolved in DMF (1 mL) and mixed with 24 µL triethylamine. Next, the TPGS-NHS and RGD DMF solution were mixed drop by drop and stirred for 24 h (25°C). The mixture was dialyzed against deionized water for purification (2 days) to remove unreacted reagents. The water was evaporated at 50°C and left to dry in a vacuum. 2.2.3 Characterization of VES-g-CSO and TPGS-RGD Samples were analyzed by Fourier transform infrared (FT-IR) spectra, elemental analysis, and proton nuclear magnetic resonance (1H-NMR) spectroscopy. FT-IR spectra of CSO, VES-g-CSO, VES, TPGS, and TPGS-RGD were recorded in KBr pellets with a FT-IR spectrophotometer (Nicolet 6700, Thermo Fisher Scientific, Waltham, USA). For 1H-NMR analysis, the samples were analyzed on a Bruker DPX300 spectrometer. VES-g-CSO and TPGS-RGD were dissolved in D2O at 25°C, and analyzed by 400 MHz NMR spectrometer (Bruker, Karlsruhe, Germany). The substitution degree of VES to VES-g-CSO was calculated using an element analyzer (CS-344 carbon/sulfur analyzer, LECO, St. Joseph, MI, USA). 2.3 Preparation of PTX@VeC/T-RGD MM The organic solvent emulsification-evaporation method was used to prepare the mixed micelles. For VES-g-CSO (VeC) micelles, 200 µL chloroform was added to the VES-g-CSO aqueous solution (10 mg VES-g-CSO in 4 mL distilled water), and the mixed solution was sonicated with a probe-type sonifier (BILON92-II DL, Shanghai China) for 5 min at 100 W with the pulse turned off for 1 s at intervals of 1 s. After that, the micelles solution was stirred on a magnetic plate at 200 rpm for 10 min, and then the speed was adjusted to 100 rpm for 20 min to ensure that the organic solvent was fully evaporated. The preparation of PTX loaded VeC (PTX@VeC) micelles and coumarin-6 labeled VeC micelles was the same as that of VeC micelles except that 1 mg PTX or 20 µg coumarin-6 was separately contained in 200 µL chloroform. For VES-g-CSO/TPGS mixed micelles (VeC/T MM), VES-g-CSO (10 mg) was dissolved in 3.4 mL distilled water, followed by addition of 0.6 mL TPGS aqueous solution (5 mg/mL). The next steps of the preparation process of VeC/T MM, coumarin-6 labeled VeC/T MM and PTX loaded VeC/T mixed micelles (PTX@VeC/T) 7



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were the same as those used for VeC micelles and PTX@VeC micelles. TPGS-RGD (0.26 mg) and TPGS (2.76 mg) in 3 mL distilled water were added into the VES-g-CSO solution to prepare the VES-g-CSO/TPGS-RGD mixed micelles (VeC/T-RGD MM), PTX loaded VeC/T-RGD mixed micelles (PTX@VeC/T-RGD MM) and coumarin-6 labeled VeC/T-RGD MM. Excess unentrapped PTX or coumarin-6 was removed using 0.22 µm filters. 2.4 Characterization of PTX@VeC/T-RGD MM 2.4.1 Particle size, zeta potential and morphology Malvern Zetasizer Nano ZS90 (Malvern Instruments, Malvern, UK) dynamic light scattering (DLS) instrument was used for the measurement of average particle size and zeta potential of the micelles. The morphology of the micelles was examined by transmission electron microscopy (TEM) (JEM-2100, Tokyo, Japan) and atomic force microscopy (AFM) (Veeco Instruments Inc, Plainview, NY, USA). 2.4.2 Drug loading coefficient (DL%) and encapsulation efficiency (EE%) A total of 20 µL of PTX-loaded micelle solution was collected and diluted with acetonitrile to determine DL and EE. The collected sample was vortexed and sonicated, and then transferred to high performance liquid chromatography (HPLC) to analyze PTX concentration.29 2.4.3 In vitro release and stability of PTX@VeC/T-RGD MM A PTX-loaded micelle solution (0.1 mg PTX contained) was sealed into a dialysis bag (MWCO = 1kDa) and submerged into 50 mL PBS (pH 7.4) containing 0.5% (w/w) tween-80 at 37°C with stirring at 100 rpm for 72 h. At the indicated time intervals, 500 µL aliquots were withdrawn, and replaced with 500 µL fresh medium. The content of PTX was determined by HPLC. All micelles were stored at 4°C, and the particle sizes were measured on days 0, 1, 2, 3, 4, 5, 6, and 7. 2.4.4 Critical micelle concentration determination The CMC of the VES-g-CSO solution with or without the addition of TPGS was determined by pyrene as the fluorescence probe30 and then recorded on a F-2500 fluorescence spectrophotometer (Hitachi High-Technologies Corp., Tokyo, Japan). All samples were excited at 335 nm. The emission wavelengths were I1 = 373 nm and I3 = 8


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385 nm. Fluorescence spectra were recorded between 350 and 400 nm. 2.5 Cellular uptake U87 MG cells were seeded in 24-well culture plates at a density of 1×104 cells per well. After incubating for 24 h (37°C), the medium in each well was refreshed with 500 µL DMEM containing free coumarin-6, coumarin-6 labeled VeC, coumarin-6 labeled VeC/T MM and coumarin-6 labeled VeC/T-RGD MM solution with a coumarin-6 (500 µg/mL) for 0.5 h or 2 h (37°C); fluorescence images were recorded immediately31. To determine whether the c(RGDfK) peptide could hinder VeC/T-RGD MM mediated endocytosis, U87MG cells were pre-incubated with free c(RGDfK) peptide (0.3 µg/mL) for 1 h before exposure to VeC/T-RGD MM. 2.6 Cytotoxicity assay U87MG cells were incubated in 96-well plates at a density of 5×103 cells per well for 24 h.The medium was replaced with DMEM containing either blank micelles-VeC micelles and VeC/T MM or PTX formulations-PTX@VeC/T-RGD MM, PTX@VeC/T MM, PTX@VeC micelles and free PTX at various concentrations, and incubated for 72 h. After the incubation, the supernatant was discarded. Then 20 µL MTT (5 mg/mL) solution was added to each well for 4 h, and absorbance was measured at 490 nm with a microplate reader (TecanSafire 2; Tecan Group Ltd., Männedorf, Switzerland). 2.7 Cell apoptosis assay U87MG cells were seeded in six-well culture plates at a density of 1×105 cells per well, cultured at 37°C for 24 h and washed with PBS twice. Cells were then incubated for 24 h with various PTX formulations (0.1 µg/mL PTX) for 24 h. After the incubation, the culture fluid was collected and the cells were washed twice with PBS. The cells were trypsinized, washed with pre-chilled PBS three times, and resuspended with PBS. After that, cells were stained using the Annexin V-FITC Apoptosis Detection kit, and a flow cytometer (FACS Calibur, BD, San Jose, CA, USA) was used to analyze cell apoptosis. 2.8 Inhibition of U87MG tumor spheroid growth U87MG cells at a density of 2×104 cells per well were seeded in 24-well plates 9



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which were pre-coated with 300 µL of 2% low melting point agarose. Seven days after seeding, the spheroids were incubated with 500 µL of serum-free DMEM containing PTX, PTX@VeC/T MM or PTX@VeC/T-RGD MM with a PTX concentration of 0.1 µg/mL. Tumor spheroids incubated with DMEM were used as controls. After various treatments, growth inhibition was evaluated by measuring the length and width of the U87MG tumor spheroids using an inverted phase microscope every day. The volume of spheroids (V) was calculated with the following equation (1)32: V=

π × length × width2 6

…………………………………………… (1)

The volume ratio of tumor spheroid (R) was calculated as equation (2): V

R = V i × 100%…………………………………………… (2) 0

Vi: the volume of U87MG tumor spheroid at the ith day after treatment; V0: the tumor spheroid volume before treatment. The inhibitory rate of tumor (IRT) on day 7 was calculated as equation (3): IRTv (%) =

Vc - Vt Vc

× 100% ………………………………… (3)

Vc: the tumor spheroids volume in the control group; Vt: the tumor spheroids volume in the treatment group on day 7. 2.9 In vivo therapeutic study and safety evaluation A total of 5×106 U87MG cells were injected subcutaneously to establish a U87MG xenograft-bearing mice model. When the tumor volume reached 40-80 mm3, the mice were randomly divided into four groups (n = 5) to receive saline, free PTX, PTX@VeC/T MM or PTX@VeC/T-RGD MM via tail intravenous injection on days 0, 2, 4, and 6 (PTX dosage: 10 mg/kg; free PTX was diluted by saline). Body weight and tumor size of mice were monitored every 2 days.33 The volume was calculated as 0.5 × length × (width)2. On day 12, the mice were sacrificed by cervical dislocation, and the tumor mass was harvested and weighed. IRT was calculated as equation (4): IRTw (%) =

Wc - Wt Wc

× 100% ……………………………………… (4)

Wc: tumor weight in the saline group; Wt: tumor weight in the treatment group. 10


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Major organs were collected for histochemistry analysis. Tissue samples were fixed and embedded. Sections were cut at 5 µm thickness with hematoxylin and eosin (H&E) staining, and visualized under a microscope (Nikon, Ti-S, Sendai, Japan). To evaluate the target effect of PTX@VeC/T-RGD MM on nascent vessels in the tumors after treatment, tumor sections were stained with DAPI and Alexa Fluor 594 anti-mouse CD31 antibody for visualization of cell nuclei and tumor angiogenesis, respectively, under a fluorescence microscope.34 2.10 Statistical analysis The results are shown as the mean and standard deviation (SD). Statistical significance was determined using ANOVA-Dunnet’s test. P values of < 0.05 were considered significant.

3. Results and discussion 3.1 Synthesis and characterization of VES-g-CSO and TPGS-RGD Figure 1A outlines the procedure used for the synthesis of VES-g-CSO. The proton peak at approximately 2.5 ppm belongs to the succinyl methylene group in VES (Fig. 2A). In the FT-IR spectrum of CSO and VES-g-CSO (Fig. 2C, D), because of the substitution of VES on the CSO backbone, the intensity of the primary amines at 3440 cm-1 was reduced. An obvious enhanced peak at 1635 cm-1 further confirmed the structure of VES-g-CSO. The yield of VES-g-CSO was 81.9%. Figure 1B describes the procedure used for the synthesis of TPGS-RGD. The signals at 2.5-3.0 ppm (Fig. S1C) corresponded to the mono succinate in SA-TPGS, and methylene appeared at 2.5-3.0 ppm (Fig. S1D) corresponding to the succinimide in NHS-TPGS. The yield of SA-TPGS and NHS-TPGS was 70.27% and 28.86%, respectively. The signals at 7.0-7.3 ppm (Fig. 2B) corresponded to the hydrogen in the benzene ring in RGD. The appearance of amide bands at 1637 cm-1 and 1552 cm-1 in the spectra of TPGS-RGD confirmed successful functionalization of TPGS (Fig. 2D). The yield of TPGS-RGD was 49.6%. These observations indicated that the VES-g-CSO and TPGS-RGD were successfully synthesized. The degree of substitution of VES, calculated by elemental analysis, was 5.21% ± 0.89%, indicating that nearly 5 of 100 11



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amino groups in the glucosamine units of CSO were substituted by VES.

Fig. 1 Synthesis of VES-g-CSO (A) and TPGS-RGD (B); the formulation mechanism of PTX@VeC/T-RGD MM (C).

12


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Fig. 2 1H-NMR (A, B) and FT-IR (C, D) of VES-g-CSO and TPGS-RGD.

3.2 Physicochemical characteristics of micelles The formulation mechanism of PTX@VeC/T-RGD MM is shown in Fig. 1C. The characteristics of blank micelles and PTX loaded micelles are shown in Table 1 and Table 2, respectively. The polydispersity index of PTX@VeC/T-RGD MM was < 0.3, as reflected in the representative particle size graph in Fig. 3C. After the insertion of TPGS, the size of PTX loaded micelles decreased to 150.2 nm, and the zeta potential decreased to 18.3 mV. This decrease may be attributed to the hydrophobic tocopherol segment in TPGS inserted into the hydrophobic shell of the micelles, which tightened the hydrophobic shell of the micelles; the hydrophilic polyethylene glycol shell may have covered the amine groups of the backbone thus concealing the partial positive charge and reducing the zeta potential value. These results were in accordance with previous reports.35,36 The DL% and EE% of PTX@VeC/T-RGD MM were 5.92% and 82.0%, respectively. The increased EE% and DL% compared with those of PTX@VeC micelles may be attributed to the solubility enhancement of TPGS. The TEM and AFM images (Fig. 3A, B) indicated that PTX@VeC/T-RGD MM were well dispersed with spherical shape. TEM images showed that the size of 13



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the mixed micelles was approximately 150 nm, which was slightly smaller than the size in DLS (Fig. 3C). The difference could be related to the dry atmosphere of TEM and the swelling condition of DLS.

Fig. 3 TEM image (A) and AFM image (B) of PTX@VeC/T-RGD MM; particle size and size distribution of PTX@VeC/T-RGD MM obtained from dynamic light scattering (C); in vitro release of PTX from micelles in PBS (pH= 7.4) (D); particle sizes of PTX loaded micelles stored at 4°C as a function of time (E) (n= 3).

Table 1 Physicochemical property of blank micelles (n = 3). Particle size Zeta potential Polydispersity Formulation (nm) (mV) index VeC micelles VeC/T MM

174.6± 5.6 116.2± 1.1

32.5± 0.3 19.4± 0.6

0.23 0.28

VeC/T-RGD MM

120.2± 0.8

16.2± 0.5

0.25

Table 2 Physicochemical property of PTX loaded micelles (n = 3). EEa (%)

DLb(%)

Particle size (nm)

Zeta potential (mV)

Polydispersity index

PTX@VeC micelles

14.3± 0.4

1.45± 0.2

196.8± 4.4

35.0± 0.1

0.29

PTX@VeC/T MM

84.1± 1.0

6.10± 0.3

158.2± 2.4

17.4± 0.6

0.20

PTX@VeC/T-RGD MM

82.0± 0.8

5.92± 0.3

150.2± 6.4

18.3± 0.4

0.24

Formulation

Note: a, EE: entrapment efficiency; b, DL: drug loading coefficient.

3.3 Critical micelle concentration determination 14


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The CMC was defined as the lowest concentration of polymer at which micelle formation was observed, and this was dependent on the nature of the polymer used. At a low CMC value, the micelles barely disintegrated and maintained integrity upon dilution.37 As shown in Fig. 4, the CMC value of VES-g-CSO solution (50.22 µg/mL) was higher than that of the VeC/T MM solution (20.14 µg/mL). Therefore, VES-g-CSO and TPGS could form micelles and resist dilution, indicating good stability in an aqueous solution. The hydrophilic corona, which was formed by polyethylene glycol segments in TPGS, provided a stabilizing interface for the drug.

Fig. 4 Plot of the fluorescence intensity ratio (I1/I3) as a function of the logarithm concentration of VES-g-CSO (A) and VES-g-CSO/TPGS (10/1, w/w) (B) solution. CMC values were related to the I1/I3 ratio of the emission spectrum profile.

3.4 In vitro drug release and stability studies Figure 3D shows the release of PTX@VeC/T-RGD MM in PBS. During the initial 30 min, all the drug loaded micelles showed burst release. After 4 h, less than 18% of the drug was released from the mixed micelles and nearly 27% of the drug was released from the PTX@VeC micelles, and this result might be related to the desorption of PTX on the surface of the micelles. Concentrations of 60.95%, 48.13% and 46.32% of encapsulated drug in PTX@VeC micelles, PTX@VeC/T MM and PTX@VeC/T-RGD MM were released after 24 h, respectively. A fraction of 75.44% of PTX was released from PTX@VeC micelles after 72 h, which may have been due to diffusion of the encapsulated drug. The release rate of PTX in PTX@VeC/T MM 15



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and PTX@VeC/T-RGD MM after 72 h was 62.17% and 60.24%, respectively. The similar diffusion rates could be related to the similar characteristic of the mixed micelles. Correspondingly, the mixed micelle groups did not show obvious changes in size within 7 days and displayed good stability because of the closed hydrophobic structure of the carrier, which contained the VES-g-CSO and TPGS (Fig. 3E). PTX@VeC micelles showed a trend towards increased size under the same conditions, and this might be the result of the poorly compacted inner core composed of VES.37 3.5 Cellular uptake Figure 5 shows representative fluorescent images of U87MG cells treated with coumarin-6 labeled VeC/T-RGD MM; the uptake of U87MG cells was time-dependent. Coumarin-6 labeled VeC/T-RGD MM showed the highest cellular uptake, induced by integrin receptor-mediated endocytosis. In addition, TPGS reduced the efflux of P-gp in cancer cells, thus promoting cellular uptake.38 After pre-treatment with free RGD peptide, the cellular uptake of coumarin-6 labeled VeC/T-RGD MM was reduced, which may be due to the competitive binding of integrin to free ligands. The quantitative evaluation of U87MG cells treated with various PTX formulations showed that the U87MG cellular uptake of PTX was time-dependent (Fig. S3), which was in accordance with the qualitative manner of cellular uptake of coumarin-6 labeled VeC/T-RGD MM.

16


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Fig. 5 U87MG cellular uptake after 0.5 h (A) and 2 h (B) of incubation with coumarin-6 labeled VeC micelles, coumarin-6 labeled VeC/T MM, coumarin-6 labeled VeC/T-RGD MM, or pre-incubation with 0.3 µg/mL of free RGD for 1 h before exposure to coumarin-6 labeled VeC/T-RGD MM was examined by fluorescent microscopy. Green: coumarin-6; bar: 100 µm.

3.6 In vitro cytotoxicity assay The MTT assay was performed to evaluate the in vitro biocompatibility of blank micelles and the cytotoxicity of different PTX formulations on U87MG cells.The blank micelles showed almost no obvious cytotoxicity at the range of 0.1-50 µg/mL (Fig. 6A). At the range of 50-1000 µg/mL, the blank micelles displayed cytotoxicity as the concentration increased. The slight cytotoxicity might be the positively charged surface of the blank micelles, for TPGS is a safe pharmaceutical excipient approved by the Food and Drug Administration. The IC50 value was 0.523 ± 0.021 µg/mL, 0.142 ± 0.008 µg/mL, 0.086 ± 0.015 µg/mL and 0.042 ± 0.003 µg/mL for PTX, PTX@VeC micelles, PTX@VeC/T MM and PTX@VeC/T-RGD MM, respectively (Fig. 6B). The increased cytotoxicity against U87MG cells observed upon the addition of TPGS might be relevant to the accelerating U87MG cellular uptake of TPGS inserted mixed micelles.36 In addition, after the addition of RGD, the PTX@VeC/T-RGD MM was the most cytotoxic of all the PTX formulations and this could be attributed to the increased endocytosis mediated by the integrin receptor.

17



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Fig. 6 Viability of U87 MG cells treated with blank micelles (A); in vitro cytotoxicity of PTX formulations against U87MG cells (B) following incubation for 72 h at 37°C (n= 3).

3.7 Cell apoptosis assay Cell apoptosis was assessed using the Annexin V-FITC staining assay. Figure 7 A, B shows that free PTX treatment did not induce a significant apoptosis increase. After the introduction of TPGS, the percentage of U87MG cells at the early and late apoptosis stages in the PTX@VeC/T MM treated group was 20.01%, which was approximately 1.71-fold higher than that in the PTX@VeC micelles group. In the RGD-conjugation group, up to 33.8% of U87MG cells were at the early and late apoptosis stages, and the PTX@VeC/T-RGD MM group showed an obvious increase in apoptosis compared with other groups at both the early and late apoptosis stages (P < 0.01). These demonstrated PTX-loaded micelles had a marked effect on inducing cell apoptosis. The addition of TPGS caused an increased number of cells to enter the apoptosis stage, and this effect was further enhanced by the decoration of RGD, which promoted the cellular uptake of micelles through integrin-mediated endocytosis39 and strengthened cell apoptosis by increasing intracellular amounts of PTX.

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Fig. 7 Quantification of PTX-induced apoptosis in U87MG cells. Cells were incubated with PTX, PTX@VeC micelles, PTX@VeC/T MM, or PTX@VeC/T-RGD MM for 24 h. Untreated cells served as controls. The pictorial distribution of cells in a single study (A), and column chart showing the average of three studies (B). ⁎P< 0.05 and

⁎⁎

indicate statistical significance levels compared with the control; #P< 0.05 and

##

@

indicate statistical significance levels compared with free PTX; P< 0.05 and

P< 0.01 P< 0.01

@@

P< 0.01

indicate statistical significance levels compared with PTX@VeC/T MM.

3.8 Inhibition of U87MG tumor spheroid growth A curative

drug

delivery

system

should ensure

the

penetration

of

tumor-associated tissues, which are usually hypoxic and avascular. Multicellular 19



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three-dimensional tumor spheroids are used to mimic the extracellular matrix of tumors in vivo.40 As shown in Fig. 8A, untreated U87MG tumor spheroids grew faster than others. Treatment with PTX@VeC/T-RGD MM led the tumor spheroids to shrink over time, with the smallest final U87MG tumor spheroid volume observed in the PTX@VeC/T-RGD MM. Figure 8B shows the changes in U87MG tumor spheroid volumes over time in the different treatment groups. The average tumor spheroid volumes of the control and PTX groups increased by approximately 3.65-fold and 3.32-fold, respectively. This slight inhibition of tumor spheroid growth inhibition could be attributed to multidrug resistance induced by PTX. In the PTX@VeC/T MM group, the tumor spheroid volume increased by approximately 2.60-fold by day 7 and the IRTv was 21.7% (P < 0.01), which might be explained by the ability of TPGS-based micelles to downregulate multidrug resistance in tumors.41 In the PTX@VeC/T-RGD MM group, tumor spheroid volume increased by 1.32-fold by day 7 and the IRTv was 49.3% (P < 0.01). The differences in tumor spheroid volumes between the groups treated with PTX@VeC/T-RGD MM and either PTX (P < 0.01) or PTX@VeC/T MM (P < 0.05) were both statistically significant, suggesting that PTX@VeC/T-RGD MM significantly enhances the inhibitory effects on U87MG tumor spheroids. After all, the higher inhibitory effect indicates the potential of PTX@VeC/T-RGD MM to improve therapeutic effects in vivo. In vivo fluorescent imaging suggested that the decoration of mixed micelles with c(RGDfK) substantially targeted them to integrin-rich U87MG tumors in vivo and reduced their nonspecific uptake by the reticuloendothelial system (Fig. S2).

20


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Fig. 8 Confocal images of U87MG spheroids after treatment with PTX, PTX@VeC/T MM, or PTX@VeC/T-RGD MM, and the control group on days 1, 3, 5, and 7 (A). Growth inhibition curves of U87MG tumor spheroids with various PTX formulations (B) (n= 3).

3.9 In vivo therapeutic study and safety evaluation Various formulations of PTX were injected intravenously into nude mice at a dosage of 10 mg/kg every other day. The tumor volumes and body weights were 21



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recorded. As shown in Fig. 9A and C, PTX@VeC/T-RGD MM treatment showed obvious tumor growth inhibition, and the IRTw values for PTX, PTX@VeC/T MM, and PTX@VeC/T-RGD MM were 31.4%, 68.7%, and 88.4%, respectively, on day 12. Mice treated with PTX@VeC/T MM and PTX@VeC/T-RGD MM did not show significant weight loss during the treatment period (Fig. 9B). The results showed that PTX@VeC/T-RGD MM had greater antitumor effects in U87MG tumor bearing mice than in the other groups. In addition, H&E staining (Fig. 9D) showed the area of necrosis in tumor tissues was larger in the PTX@VeC/T-RGD MM group than in the other groups. By contrast, PTX and PTX@VeC/T MM treated tumor tissues showed many viable tumor cells, indicating that the therapeutic efficacy of PTX@VeC/T-RGD MM was greater than that of PTX and PTX@VeC/T MM against U87MG tumors. Tumor vessels were stained with Alexa Fluor 594-conjugated anti-CD31 antibody. The polyethylene glycol moiety of TPGS and RGD, which localizes to the outer layer, can prolong blood circulation times and promote tumor-targeting, which may have contributed to the better therapeutic efficacy of PTX@VeC/T-RGD MM. In Fig. 9E, the tumor vessels are stained green in the saline group. A decreased amount of vessels in the PTX@VeC/T-RGD MM group compared with those in the other three groups reflects the inhibitory effect of RGD decorated PTX-loaded mixed micelles on nascent vessels. In addition, there is an affinity between the RGD peptide and nascent vessels

expressing

integrin

αvβ3

receptors,41

which

may

have

facilitated

PTX@VeC/T-RGD MM accumulation on nascent vessels, resulting in an anti-angiogenesis effect. As a result, the introduction of RGD into mixed micelles could contribute to cutting off the nutrient supply to the tumor and improving the therapeutic efficacy. H&E stained images of major organs (Fig. 10) revealed no visible damage to these tissues in the different treatments. Taken together, the results of in vivo studies indicated that PTX@VeC/T-RGD MM had excellent therapeutic efficacy with low toxicity to normal tissues.

22


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Fig. 9 Tumor growth curves of the different groups treated as indicated (A); body weight changes of mice during the treatment period (B) (n= 5). Photographs of tumors from each group after treatment (C) and images of hematoxylin and eosin-stained tumor tissue sections processed from subcutaneous tumor-bearing mice after treatment (D). In vivo tumor distribution of various formulations of PTX (E). Blue: DAPI stained cell nuclei. Green: CD31 stained tumor angiogenesis. Original magnification: ×20.

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Fig. 10 Histochemistry analysis of major organ sections stained with hematoxylin and eosin in various groups. Original magnification: ×20.

4. Conclusion In the present study, VES-g-CSO and TPGS-RGD were synthesized and applied to

prepare

mixed

micelles

encapsulating

PTX

by

the

organic

solvent

emulsification-evaporation method. The PTX@VeC/T-RGD MM, which showed high drug loading capacity (5.92%), were 150.2 nm in diameter and had a low zeta potential (18.3 mV),possessing good stability over 7 days. The CMC value for VES-g-CSO/TPGS was as low as 20.14 µg/mL. The PTX@VeC/T-RGD MM showed sustained-release properties in vitro. Increased cellular uptake in U87MG cells and decreased cell viability were associated with PTX@VeC/T-RGD MM. PTX@VeC/T MM and PTX@VeC/T-RGD MM markedly induced apoptosis in U87 MG cells. A tumor spheroid penetration study further provided evidence that PTX@VeC/T-RGD 24


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MM enhanced tumor spheroid inhibition. The affinity of the PTX@VeC/T-RGD MM for the integrin receptor overexpressed in U87MG cells and nascent vessels contributed to their increased therapeutic effect in vivo and safety to normal tissues. Taken together, these demonstrated that VeC/T-RGD MM is an ideal nanocarrier for hydrophobic chemotherapeutic drug delivery.

Supporting Information 1

H-NMR spectra of VES (Fig. S1A), TPGS (Fig. S1B), TPGS-SA (Fig. S1C) and

TPGS-NHS (Fig. S1D), in vivo NIR imaging analysis and dissected organs of subcutaneous-U87MG tumor-bearing mice (Fig. S2), and quantitative evaluation of various PTX formulation uptake in U87MG cells (Fig. S3).

Acknowledgments This work was sponsored by the National Natural Science Foundation of China (No.81503021, 81603502) and the Fundamental Research Funds for the Central Universities (22A201514055 ECUST). Abbreviations VES: vitamin E succinate CSO: chitosan oligosaccharide TPGS: D-alpha-tocopheryl polyethylene glycol 1000 succinate RGD: arginine-glycine-aspartic acid peptide PTX: paclitaxel TPGS-RGD: RGD-conjugated D-alpha-tocopheryl polyethylene glycol 1000 succinate VES-g-CSO: vitamin E succinate-grafted-chitosan oligosaccharide VeC: VES-grafted-CSO micelles VeC/T MM: VES-grafted-CSO/TPGS mixed micelles VeC/T-RGD MM: VES-grafted-CSO/TPGS-RGD mixed micelles PTX@VeC: PTX loaded VES-grafted-CSO micelles PTX@VeC/T MM: PTX loaded VES-grafted-CSO/TPGS mixed micelles PTX@VeC/T-RGD MM: PTX loaded VES-grafted-CSO/TPGS-RGD mixed micelles SA: succinic anhydride NHS: N-hydroxysuccinimide 25



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DMAP: 4-dimethylaminopyridine DCC: N,N-dicyclohexylcarbodiimide EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide TEA: triethylamine

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Abstract Graphic

30


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1H-NMR (A, B) and FT-IR (C, D) of VES-g-CSO and TPGS-RGD. 597x394mm (80 x 80 DPI)


Vitamin E Succinate-Grafted-Chitosan ... - ACS Publications

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