ATRP for Tumor-Targeted - American Chemical Society

Subscriber access provided by La Trobe University Library

Article

pH/reductant dual-responsive core-crosslinked micelles via facile in-situ ATRP for tumor-targeted delivery of anticancer drug with enhanced anticancer efficiency Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00241 • Publication Date (Web): 05 Jul 2016 Downloaded from http://pubs.acs.org on July 10, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

pH/reductant dual-responsive core-crosslinked micelles via facile in-situ ATRP for tumor-targeted delivery of anticancer drug with enhanced anticancer efficiency Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: Anticancer drugs cause severe side effects on normal tissues and organs due to their nonspecific delivery. Thus tumor-targeting delivery of anticancer drugs remains a serious challenge in chemotherapy. Here a facile strategy was established for the pH/reductant dualresponsive core-crosslinked (CCL) micelles for tumor-targeted delivery of anticancer drug, via in-situ atom transfer radical polymerization (ATRP). In the in vitro controlled release experiments with doxorubicin (DOX) as a model drug, the premature drug leakage rate was only 13.4% in the physiological medium within 36 h, while the cumulative release rate in the stimulated tumor microenvironment reached 78.7%, demonstrating the excellent tumor microenvironment responsive controlled release behavior upon acidic media with high GSH level. As a folate receptor (FR)-mediated targeting drug delivery system (DDS), the micelles showed excellent cytocompatibility, and enhanced anticancer efficiency after loading of DOX, compared with free DOX.

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 28

Keywords: Drug delivery system; tumor-targeting; core-crosslinked micelles; controlled release; tumor microenvironment-responsive

INTRODUCTION As the main adjuvant way accompanying surgery for present clinical cancer treatment, chemotherapy is much to be desired because that almost all the anticancer drugs used in the chemotherapy course cause severe side effects on normal tissues and organs due to their nonspecific delivery.1,2 To meet this challenge, intelligent drug delivery system (DDS) has attracted more and more attention because of their potential tumor-targeted delivering function,3,4 including the passive targeting effect via the enhanced permeability and retention (EPR) effect of the nanoscaled DDS,5 and/or the active targeting effect by precisely modulating the drug release in different tissues,6 or conjugating with tumor-cell-specific small-molecule ligands.7 On the other side, the bioavailability of anticancer drugs after oral administration is usually low because the reduced absorption, and intravenous administration of these drugs is challenging and requires a formulation with organic solvents and classical surfactants (e.g. the Taxol formulation of paclitaxel from Bristol-Myers Squibb).8 Fortunately, solubilization of hydrophobic drugs in the core of micelles can overcome this problem. Polymeric micelles with hydrophobic cores and hydrophilic shells, formed by the self-assembly of the block-copolymers with hydrophilic and hydrophobic blocks in an aqueous environment, show several advantages over other nanosized drug delivery systems, such as a smaller size, which is important for, e.g., percutaneous lymphatic delivery or extravasation from blood vessels into the tumor tissue.9 Besides the micellar drug solubilization effect, targeting ligands can be attached to the micelles in order to specifically recognize and bind to the receptors overexpressed in tumor cells,10 and pH-11, or thermo-sensitive12 block copolymers allow for triggered drug release.


2

Page 3 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Burst release is one of the challenges limiting the clinical application of block copolymer micelles for targeted delivery of lipophilic anticancer drugs. It’s generally assumed that burst drug release in blood circulation is mainly due to blood dilution and subsequent micelle disassembly after intravenous administration. Recently, the crosslinked polymeric micelles have been developed to prevent premature disintegration and subsequent burst drug release.13 Especially for those crosslinked with the bioreducible disulfide bonds, the disulfide crosslinkages could efficiently prevent the dissociation of the micelles into unimers upon extensive dilution, while the payload was released at a high rate under intracellular-like conditions (e.g., pH 5 with the presence of glutathione (GSH)), showing reductant-triggered drug release.14-18 In these works, the functional copolymers were synthesized at first and then self-assembled and crosslinked with ideal physiological stability to reduce drug leakage. In this present work, a novel functional core-crosslinked (CCL) micelles was in-situ synthesized via atom transfer radical polymerization (ATRP) of tert-butyl acrylate (tBA) and N,N'-bis(acryloyl)cystamine (BACy) with folate-functionalized macroinitiator, followed by hydrolysis. Here, folate group was introduced to achieve the folate receptor (FR)-mediated endocytosis,19 and the disulfide crosslinking bond was used to make the CCL reductant triggering. After the tBA units were hydrolyzed, plentiful carboxylic acid groups were produced into the hydrolyzed core-crosslinked (HCCL) micelles for the drug loading and subsequent pHresponsive controlled release (Scheme 1). The combination of these approaches will further improve specificity and efficacy of micelle-based drug delivery and brings the development of a ‘magic bullet’ a major step forward.


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 28

Scheme 1. Preparation of the multi-functional CCL micelles via in-situ ATRP and their intracellular release behavior.

EXPERIMENTAL SECTION Materials and reagents. Poly(ethylene glycol) (HO-PEG-OH, Mn=2000), 2-bromoisobutyryl bromide (97.0%) and folic acid (FA, A.R. grade) was got from Beijing Chemical Works ,TCI and Tianjin Guangfu Fine Chem. Res. Inst., respectively. Triethylamine (TEA, AR, Tianjin Rionlon Pharmaceutical Chemical Co., Ltd.) was dried by stirred with CaH2. Cuprous bromide (CuBr, AR, Tianjin Guangfu Fine Chem. Res. Inst) was stirred with glacial acetic acid 12 h and then washed with anhydrous ether, anhydrous ethanol


4

Page 5 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and acetone. Tert-butyl acrylate (tBA, 99%, Aladdin) was distilled under reduced pressure after treated

with

CaH2.

Dicyclohexylcanbodiimide

(DCC,

C.P.

grade)

and

1,1,4,7,7-

pentamethyldiethylenetriamine (PMDETA, >98%) were purchased from Shanghai Kefeng Chem. Rea. Co., Ltd. and Tokyo KASET Kogyo. Co. Ltd., respectively. Other reagents were of analytical grade and used directly without any further treatment. Deionized water was used throughout the experiments.

Synthesis of macroinitiator FA-PEG-PtBA-Br. The diblock copolymer FA-PEG-PtBA-Br was prepared as macroinitiator by three steps. Firstly, the HO-PEG-Br was synthesized by the typical procedure reported previously:20 300 mL of dried toluene containing 20g HO-PEG-OH (0.01 mol) was treated by vacuum distillation at 70°C for the purpose of eliminating water. After 50 mL toluene had been distilled out, TEA (2.98 mL, 0.02 mol) was added into the reacting mixture and the temperature was cooled to 0 °C. Then, 2-bromoisobutyl bromide (1.23 mL, 0.01 mol) was added slowly within 0.5 h. After stirring for 8 h at room temperature, most of toluene was removed by reduced pressure distillation and the crude product was obtained by precipitating in cold ether. Then the crude product was dissolved in NaHCO3 saturated aqueous solution and then extracted with dichloromethane for three times. Most solvent was removed by reduced pressure distillation after treating with anhydrous MgSO4 to remove any trace water, the white solid emerged by precipitation with cold ether, and dried under vacuum overnight. FA-PEG–Br was synthesized by the following procedure:21 Folic acid (0.6628 g, 1.5 mmol) was dissolved into 10 mL dimethyl sulfoxide (DMSO) by ultrasonic and stirring. For preactivating folic acid, DCC (0.3080 g, 1.5 mmol) was chosen as coupling agent. The mixture was stirred 4 h at room temperature and 10 mL DMSO containing dehydrated HO-PEG-Br (2 g, 1 mmol) and 4-dimethylamino pyridine (DMAP, 12.21 mg, 0.1 mmol) was added dropwise into


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 28

the reaction within 0.5 h. The polymerization was conducted with stirring at 50 °C for 12 h and then the system was transferred into dialysis tubes for 2 d to remove the remained materials, and the desired product was obtained by vacuum filtration and drying under vacuum overnight. The diblock copolymer FA-PEG-PtBA-Br was prepared via the atom transfer radical polymerization (ATRP) procedure with FA-PEG-Br as macroinitiator.22 FA-PEG-Br (1.0 g, 0.5 mmol), tBA (8 mL, 50 mmol), PMDETA (0.0684 mL, 0.33 mmol) and cyclohexanone (3 mL) were added to a totally dried reaction flask. The system was treated with three freeze-pump-thaw cycles in order to remove any trace oxygen. Then CuBr (73 mg, 0.5 mmol) was added into flask. The reacting mixture was stirred at 70 °C for 12 h. The resulting mixture was diluted into 5 mL CH2Cl2. The residual catalyst was removed by passing neutral alumina column. The copolymer was collected by precipitation in cold methanol–H2O (1:4, v/v) solution, and drying under vacuum overnight.

Preparation of core cross-linked micelles. The core cross-linked (CCL) micelles were synthesized via ATRP copolymerization with diblock copolymer FA-PEG-PtBA-Br as macroinitiator, BACy as cross-linking agent, and tBA as monomer. The reagent molar ratio was [FA-PEG-PtBA-Br]:[tBA]:[BACy] = 1:100:5. 0.10 mmol FA-PEG-PtBA-Br and corresponding amounts of tBA and BACy were added into a mixed solution of 4 mL methanol and 1 mL cyclohexanone. After stirring at 70 °C for 12 h, the crude product was dialyzed against THF for 2 days in order to remove any remained monomer or macroinitiator, and water for 2 days to completely remove Cu catalyst. Finally, the CCL micelles were obtained by vacuum filtration.


6

Page 7 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hydrolysis of the CCL micelles. 4 mL CH2Cl2 containing 1 mL trifluoroacetic acid was used to hydrolyze the ester groups in the CCL micelles (1 g).21 The mixture was stirred in the dark for 24 h and the hydrolyzed CCL (HCCL) micelles were obtained after remove all solvent.

DOX-loading and in vitro controlled release. The HCCL micelles (10 mg) were dispersed in 4 mL DOX solution (1 mg/mL) with an ultrasonic oscillator. Then the dispersion was adjusted to neutral condition, and stirred for 48 h in the dark at room temperature. Afterward, the mixture was separated by centrifugation at 12000 rpm for 10 min. Supernatant solution was analyzed by UV spectrophotometer in order to calculate the drug loading capacity and efficiency while the precipitate was used for further evaluation of the release behavior. 5 mL PBS buffer solution (7.4 or 5.0, with or without reducing agent) containing 10 mg DOXloaded HCCL micelles was added into a dialysis tube (molecular weight cut off 14000), then the dialysis tube was transferred into 120 mL corresponding buffer solutions at 37.5°C and shaken in a table concentrator. 5 mL of the solution was taken out in a certain time gradient for the measurement of the DOX concentration by UV spectrophotometer, while fresh 5 mL corresponding buffer solutions added in immediately in order to keep the solution volume constant. The DOX concenteations at different releasing times were used to analyze the cumulative release.

In vitro cytotoxicity and cellular uptake. MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide] assay was used to evaluate the biocompatibility of the HCCL micelles in SKOV3 cells. Cells (1×104 cells/well) were injected into the medium containing 10% fetal bovine serum (FBS), then the aqueous HCCL dispersion (100 µL, with or without DOX) was added into each well and cultivated at condition of 5% CO2 and 37 °C for 24 h. 20 µL MTT


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 28

solution (5 mg/mL) then injected into each well and cultivated 4 h. 150 µL DMSO was added into each well after the culture solution was removed. The cell relative activity was measured using Thermo Scientific Multiskan Go at 570 nm. The cellular uptake behavior was analyzed by confocal laser-scanning microscopy (CLSM) using SKOV3 cells and Hoechst 33258. Cells which dyed by Hoechst 33258 and incubated with the DOX-loaded HCCL (20.0 µg/mL) were injected in the medium containing 100 µL of PBS for 12 h. The location of intracellular fluorescence was traced with the excitation wavelengths of 480 nm for DOX and 405 nm for Hoechst 33258.

Analysis and characterization. Chemical structures of FA-PEG-Br and FA-PEG-PtBA-Br were characterized by 1H-NMR spectra (Bruker 400 MHz spectrometer), with dimethyl sulfoxide-D6 and CDCl3 as solvent, respectively. FT-IR spectra were obtained from Nicolet 360 FTIR spectrometer (wavenumber range 4000400 cm-1). The morphology of the micelles was analyzed by transmission electron microscopy (TEM) analysis (JEM-1200EX). Hydrodynamic diameter and distribution of the micelles were analyzed by dynamic light scattering (DLS) which got from a high-performance particle sizer. Drug-loading capacity and release behavior were calculated from the UV spectrophotometer (TU-1901) analysis.

RESULTS AND DISCUSSION Synthesis and characterization of FA-PEG-PtBA-Br. 1H NMR spectrum was used to analyze the structure of the block copolymer. In the spectrum of HO-PEG-Br, the chemical shifts at 3.6


8

Page 9 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and 1.4 ppm were due to the protons in the -OCH2CH2O- and -C(CH3)2Br groups, respectively. Their signal area ratio was 27:1, near to the theoretical value of the single-end-functiomalized PEG (30:1), indicating the successful synthesis of HO-PEG-Br. After modifying the other end group with FA, the chemical shifts at 8.7 ppm (pteridine proton) and around 7.0 ppm (aromatic protons) appeared. The signal area ratio of the chemical shifts at δ 8.7 and 1.4 ppm was 1:5.3. It was also near the theoretical value (1:6) of the target product FA-PEG-Br, the double-endfunctionalized PEG with one FA end group and one 2-bromoisobutyrate end group.

Figure 1.1 H NMR spectrum of HO-PEG-Br, FA-PEG-Br, and FA-PEG-PtBA-Br.


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 28

Then the macroinitiator FA-PEG-PtBA-Br was synthesized using ATRP method with the reagent ratio set at [FA-PEG-Br]:[tBA] =1:100. As shown in Figure 1, the chemical shift at δ 8.7 ppm meant the pteridine proton while the chemical shifts at δ 6.7 and 7.4 ppm were due to the aromatic protons in FA, revealing the existence of FA in the coploymer. Furthermore, the chemical shift at δ 2.4 ppm indicated the presence of γ-CH2 in glutamic acid, which strengthened the above conclusion. The existence of PEG was illustrated by the chemical shift at δ 3.6 ppm of the protons in -OCH2CH2O-. Also, the chemical shift at δ 1.5 ppm was attributed to the -CH3 protons in the tert-butyl group of PtBA. All these data demonstrated that the macroinitiator FAPEG-PtBA-Br has been synthesized sucessfully. Also, it can be calculated from the signal area ratio of the chemical shifts at δ 1.5 ppm and δ 3.6 ppm that about 74 tBA units had been polymerized with one macroinitiator, meaning the molecular weight of 1.2×105.

Preparation and characterization of CCL micelles. The CCL micelles were synthesized with the reagent molar ratio of [macroinitiator]:[tBA]:[BACy] = 1:100:5. From the transmission electron microscopy (TEM) image (Figure 2a), it was easy to see that the CCL micelles exhibited the mophorlogy of solid microspheres with an average particle size of 180 nm. This is resulted from the factor that tert-butyl acrylate (tBA) monomers were crosslinked as hydrophobic blocks with BACy. As a result, the unique structure would lead to excellent stability upon extensive dilution and potential pH-redox responsive capability. The dispersibility of these CCL micelles was determined by dynamic light scattering (DLS). As shown in Figure 3a, the CCL micelles owned excellent dispersibility with average hydrodynamic diameter (Dh) of 220 nm and a narrow hydrodynamic diameter distribution due to their surface PEG brushes. The hydrodynamic diameter from DLS analysis was bigger than that from the TEM analysis because of the extension of the surface PEG brushes in water.


10

Page 11 of 28

(a)

(b)

Figure 2. TEM images of the CCL micelles (a) and the HCCL micelles (b).

100

100

80

80

60

Intensity

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


40 20 0 150

60 40 20

200 Dh (nm)

250

300

0 210

240

270

300

Dh (nm)

(a)

(b)


11

Page 12 of 28

700

30

600

25

500

20

400

15

300

10

200

5

Scattering Intensity (kcps)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Dh (nm)


0

100 3

4

5

6

7

8

pH

(c) Figure 3. Typical hydrodynamic diameter and distribution of the CCL micelles, (a) the HCCL micelles (b), and average hydrodynamic diameter (Dh) of the HCCL micelles and scattered light intensity at different the pH values (c).

Preparation and characterization of HCCL micelles. The hydrolyzed core-crosslinked (HCCL) micelles were obtained by hydrolysis of the core-crosslinked (CCL) micelles with trifluoroacetic acid. As a result, the crosslinked PtBA cores were turned into the crosslinked PAA cores, which could be learned from the FT-IR spectrum (Figure 4). The well-defined characteristic absorbance peaks of the carbonyl in ester group (-COOC(CH3)3) at around 1730 cm-1 (stretching vibration) and two peaks at 1454 cm-1 and 1367 cm-1 of the symmetrical deformation vibration of the tertiary butyl groups of tBA units disappeared,23 while a C=O stretch vibration of carboxylic acid appeared at 1690 cm-1.


12

Page 13 of 28

100 Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


90 80 70 60 50

CCL HCCL

4000 3500 3000 2500 2000 1500 1000

500

-1

Wavenumbers (cm )

Figure 4. FT-IR spectra of the CCL micelles and the HCCL micelles.

The particle size of the HCCL micelles remained 180 nm of the CCL micelles (Figure 2b), however their morphology became more regular and their surface became smoother. Most importantly, the aggregates in the CCL micelles disappeared after hydrolysis. This is resulted from the rearrangement of the PAA blocks in the crosslinked cores, which could be swollen in water. After hydrolysis, the interparticle chain entanglement on the surface of the micelles, which caused the aggregates of the CCL micelles, disintegrates as water soluble polymer brushes. When drying in the sampling procedure for TEM analysis, both the crosslinked cores and their surface polymer brushes shrunk to demonstrate the uniform microspheres with narrow particle size distribution. Furthermore, their Dh increased to 260 nm from 220 nm the CCL micelles (Figure 3b) because of the swelling of the crosslinked PAA cores, while remaining the outstanding dispersibility with narrow distribution.


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 28

The pH-responsive capability of the HCCL micelles was evaluated by DLS technique. The Dh increased when the media pH value increased from 3 to 8 (Figure 3c). It kept a smaller value when pH value at 3-4 while tremendous increment occurred when the pH value was higher than 4. This should be due to the pKa value of the carboxylic acid groups in acrylic acid of 4.30.24 At pH higher than 4.30, the carboxylic acid groups deprotonate into anionic carboxylate groups. The electrostatic repulsive force between the anionic carboxylate groups resulted into the further extension of the micelles,21 as well as the Donnan osmotic pressure.25 Besides, the change in scattered light intensity also illustrated the variation of particle size indirectly. The reductant-responsive characteristic of the HCCL micelles was also investigated by dispersing the micelles in different media (pH 7.4 with 10 µM GSH mimicking the normal physiological medium or pH 5.0 with 10 mM GSH mimicking the tumor microenvironment) for 36 h. In the analysis, a dispersion of the HCCL micelles in deionized water with concentration of 1 mg/mL was diluted into 0.2 mg/mL by the two kinds of PBS solutions, respectively. Then the light transmittance of the dispersion was measured by UV spectrophotometer (299 nm). The results were compared in Figure 5. At the initial stage, the transmittance of the dispersion in pH 7.4 and 10 µM GSH was higher than that in pH 5.0 and 10 mM GSH, maybe due to that the surface PEG brushes, which curl up in the media with higher ionic strength (pH 5.0 and 10 mM GSH), but extend in the media with lower ionic strength (pH 7.4 and 10 µM GSH). As time goes on, the transmittance in the medium of pH 5.0 and 10 mM GSH increased obviously, especially for the first 12 h, indicating that the HCCL micelles had been de-crosslinked into water soluble blocks by cleaving the disulfide crosslinking linkage.26 However, that in the medium of pH 7.4 and 10 µM GSH increased slightly, meaning the better stability in the stimulated normal physiological medium.


14

Page 15 of 28

pH=5.0 and 10 mM GSH pH=7.4 and 10 µM GSH

44

Transmittance (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


42

40

38 0

10

20

30

40

Time (h) Figure 5. Transmittance of the HCCL micelles dispersions in different stimulated media.

To evaluate the reductant-triggered disassembly of the HCCL micelles, they were dispersed into an aqueous solution of pH 5.0 with 10 mM GSH mimicking the tumor microenvironment. As shown in Figure 6, as time increased, the amount of the HCCL micelles with Dh of about 250 nm reduced. The Dh was slightly smaller than that in Figure 3b, due to the shrinking of the HCCL in the acidic medium. In presence of GSH, the disulfide crosslinking structure in the HCCL micelles were destroyed, therefore the HCCL micelles disassembled into the water soluble polymer. After 10 h, there were only polymer blocks (size less than 100 nm) and their aggregates (size more than 400 nm), revealing the complete disassembly of the HCCL micelles.


15


100

80

80 Intensity

100

Intensity

60 40 20

60 40 20

0 0

50

100

150

200

250

300

0

350

50

100

150

Dh (nm)

200

250

300

350

400

Dh (nm)

(a)

(b)

100 80 Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 28

60 40 20 0 0

100

200

300

400

500

600

Dh (nm)

(c) Figure 6. Typical hydrodynamic diameter and distribution of the HCCL micelles in the simulated tumor microenvironment (pH 5.0 with 10 mM GSH) for 3 h (a), 5 h (b), and 10 h (c).

DOX-loading and in vitro release. The drug-loading possibility and capability of the HCCL micelles were evaluated with DOX as a model drug. At neutral medium, the carboxylic acid groups in the PAA blocks ionize into carboxylate anions (Figure 7), while DOX protonates as cation (pKa of 8.2527). So DOX was loaded onto the HCCL micelles via electrostatic interaction.


16

Page 17 of 28

In the case, the drug loading capacity (DLC) and loading efficiency (DLE), denoted as the mass ratios of the loaded DOX and micelles, and the loaded DOX and feed DOX respectively, were calculated to be 27.8% and 69.5% respectively from the UV spectrophotometer analysis.

-25 Zeta Potential (mV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-30 -35 -40 -45 -50 4

5

6 pH

7

8

Figure 7. Zeta potential of the HCCL micelles in different pH media.

The in vitro drug release behavior of the DOX-loaded HCCL micelles was evaluated in different stimulated media. Figure 8a shows the effect of the media pH on the cumulative release without any reductant. It could be seen that the drug release rate was faster at pH 5.0 than pH 7.4 with the cumulative releases of 18.8% and 9.7% within 36 h, respectively. The higher cumulative release in the acidic media was due to that the electrostatic interaction between the micelles and DOX was weakened in the acidic media.28 Furthermore, the DOX dissolubility in acidic media is higher than that in neutral or basic media. The result revealed the pH-responsive controlled release property of the HCCL micelles. The DOX concentration in the dialysates reached equilibrium with the remained DOX in the HCCL after 36 h, so the cumulative release did not increase obviously with prolonging the releasing time.


17


a

Cumulative Release (%)

20 15 10

pH=5.0 and no GSH or DTT

5

pH=7.4 and no GSH or DTT

0 0

10

20

30

40

50

60

70

80

35

40

Time (h)

b

80 Cumulative release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 28

60

pH=5.0 and 10 mM GSH pH=5.0 and 10 mM DTT pH=7.4 and 10 µM GSH

40

pH=7.4 and 10 µM DTT

20 0 0

5

10

15 20 25 Time (h)

30

Figure 8. In vitro DOX release profiles under different conditions.

The different reductant levels in the normal tissue and tumor tissue is another obvious physiological difference besides pH value. It has been reported that the reductant level in tumor tissue is about 100-1000 times higher than that in the normal tissue.29 So the reductantresponsive DDS could also realize the tumor microenvironment-responsive “on-demand”


18

Page 19 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


anticancer drug delivery.26 This is also the aim to introduce the disulfide bond crosslinking structure of the micelles by using BACy. Figure 8b shows the release behavior in different pH values and reducing agent concentrations. Under acidic condition (pH=5.0), drug release with 10 mM GSH was more efficient than that without GSH. Within first 10 h, the former gave a higher DOX cumulative release (57.6%) while only 9.5% for the latter, and these data reached 78.7% and 18.8% within 36 h, respectively. The similar phenomena occurred in the in vitro drug release with 10 mM DTT, which manifested as 24.4% within 10 h and 33.0% within 36 h. Reducibility of GSH and DTT should be responsible for these situations. The disulfide bonds in the HCCL micelles were destroyed by the reducing agents, therefore the HCCL micelles decomposed into water soluble polymer, as discussed above (Figure 5). It is beneficial to the drug diffusion from the DDS. The difference between the cumulative release rates with GSH and DDT at the same concentration was resulted from their different structure. The amino group-containing GSH could displace the electrostatic interaction between –COO- groups in PAA blocks and –NH3+ group in DOX in the acidic media.30 Also, the positively charged amino group in GSH also produce a repulsive effect to DOX, thus the DOX release is accelerated, exhibiting a “synergistic effect”. Meanwhile, the release behavior under pH 7.4 media with 10 µM GSH or DTT were chosen on basis of the actual physiological environment of cells. Within initial 10 h, the DOX cumulative release rates were 9.6% and 7.0%, respectively. The final cumulative release rates came to only 13.4% and 10.7% within 36 h. This meant most of the loaded DOX was reserved because of the better stability of the HCCL micelles in such media (Figure 5). The in vitro DOX release experiments showed that the HCCL micelles could efficiently transport DOX molecule to the targeted site with minimal drug leakage in physiological medium. Therefore, it is expected to


19


enhance the anticancer efficiency of the anticancer drugs as well as reduce their side effect on the normal tissues.

DOX-loaded HCCL micelles HCLL micelles Free DOX

100

Cell viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 28

80 60 40 20 0 0

10

20

30

40

50

Dosage (µg/mL)

Figure 9. Viability of SKOV3 cells incubated with the DOX-loaded HCCL micelles, HCCL micelles and free DOX.

Cytotoxicity and cellular uptake. Assessment of the in vitro cytotoxicity of the HCCL micelles was executed using SKOV3 cells. Figure 9 explains the cell viability under three different conditions. At the concentration range of 5 µg/mL to 40 µg/mL, the free HCCL micelles exhibited great nontoxic property with high cell viability in the range of 98.2-94.1%. At the same concentration range, the viability of cells incubated with the DOX-loaded HCCL micelles was 48.1% to 35.3%, demonstrating obvious cytotoxicity. Meanwhile, free DOX exhibited strong toxic property to the cells, which proved by lower cell viability—45% to 7%. Considering the DLC of 27.8% and the cumulative release rate of 2.77% in 4 h (Figure 8a), the DOX concentration in the incubating fluid was much lower with the DOX-loaded HCCL micelles in


20

Page 21 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the 4 h of incubation, compared with the case with free DOX. So it could be concluded that the anticancer efficiency of DOX had been distinctly enhanced by the proposed FR-mediated targeting HCCL micelles, indicating their promising applications to overcome multi-drug resistance in tumor treatment.31

(a)

(b)

(c)

Figure 10. CLSM images of SKOV3 cells: (a) dyed by Hoechst, (b) DOX in cells and (c) merged images.

The cellular uptake of the DOX-loaded HCCL micelles was performed by confocal laserscanning microscopy (CLSM), as shown in Figure 10. The blue spots in Figure 10a meant the location of cell nuclei which were expected to receive DOX, while the red spots, attributed to the DOX fluorescence in Figure 10b, demonstrated that DOX had accumulated in the targeted sites after 12 h of incubation. The merged image (Figure 10c) clearly demonstrated that all the cell nuclei had been overlapped with DOX, showing the purple color. The results indicated that the loaded DOX had been successfully released and accumulated in the cell nuclei.32

CONCLUSIONS In summary, novel core cross-linked (CCL) micelles were synthesized via ATRP for tumortargeting delivery of anticancer drug with FA-PEG-PtBA-Br copolymer as macroinitiator and


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

BACy as crosslinker. After hydrolysis of the tBA units into AA units, the hydrolyzed CCL (HCCL) micelles with folate receptor (FR)-mediated targeting and pH/reductant dual-responsive controlled release performance were obtained with particle size of 180 nm. The drug loading capacity of 27.8% was achieved with DOX as model drug. The in vitro release experiments showed a low premature drug leakage rate of 13.4% under pH 7.4 with 10 µM GSH mimicking the normal physiological medium, while a high cumulative release rate of 78.7% under pH 5.0 with 10 mM GSH mimicking the tumor microenvironment. The cytotoxic experiments showed that the free HCCL micelles were nearly non-cytotoxic, while obvious cytotoxic after loading of DOX. And the DOX-loaded HCCL micelles could be uptaken by SKOV3 cells via the FRmediated endocytosis, and then disintegrated to release DOX responding to the tumor microenvironment (acidic media with high GSH level). The release DOX was accumulated in the cell nuclei from the CLSM analysis. Most importantly, the HCCL micelles were found to further enhance cytotoxicity in SKOV3 cells because of their better on-demand drug release capability of pH/reductant dual-responsive. These results suggest the FR-mediated targeting and pH/reductant dual-responsive micelles via in-situ self-assembly in ATRP have promising application to overcome multi-drug resistance in tumor treatment.

AUTHOR INFORMATION Corresponding Author. * Corresponding Author. Tel./Fax: 86 0931 8912582. Email: [email protected]. Notes. The authors declare no competing financial interest.

ACKNOWLEDGMENTS


22

Page 23 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


This project was granted financial support from the National Nature Science Foundation of China (Grant no. 20904017) and the Program for New Century Excellent Talents in University (Grant no. NCET-09-0441).

REFERENCES (1) Beisecker, A.; Cook, M. R.; Ashworth, J.; Hayes, J.; Brecheisen, M.; Helmig, L.; Hyland, S.; Selenke, D. Side effects of adjuvant chemotherapy: Perceptions of node-negative breast cancer patients, Psycho-oncology 1997, 6, 85–93. (2) Lima-Tenório, M. K.; Pineda, E. A. G.; Ahmad, N. M.; Fessi, H.; Elaissari, A. Magnetic nanoparticles: In vivo cancer diagnosis and therapy, Int. J. Pharm. 2015, 493, 313–327. (3) Fang, Z.; Wan, L.Y.; Chu, L. Y.; Zhang, Y. Q.; Wu, J. F. 'Smart' nanoparticles as drug delivery systems for applications in tumor therapy, Exp. Opin. Drug Deliv. 2015, 12, 1943–1953. (4) Ahmed, N.; Fessi, H.; Elaissari, A. Nanoparticles for cancer from diagnosis to theranostics, Drug Discov. Today 2012, 17, 928–934. (5) Nichols, J. W.; Bae, Y. H. EPR: Evidence and fallacy, J. Control Release 2014, 190, 451– 464. (6) Cheng, R.; Meng, F. H.; Deng, C.; Zhong, Z. Y. Bioresponsive polymeric nanotherapeutics for targeted cancer chemotherapy, Nano Today 2015, 10, 656–670. (7) Srinivasarao, M.; Galliford, C. V.; Low, P. S. Principles in the design of ligand-targeted cancer therapeutics and imaging agents, Nature Rev. Drug Discov. 2016, 14, 203–219. (8) Oerlemans, C.; Bult, W.; Bos, M.; Storm, G.; Nijsen, J. F. W.; Hennink, W. Polymeric Micelles in Anticancer Therapy: Targeting, Imaging and Triggered Release, Pharm. Res. 2010, 27, 2569–2589.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 28

(9) Trubetskoy, V. S. Polymeric micelles as carriers of diagnostic agents, Adv. Drug Deliv. Rev. 1999, 37, 81–88. (10) Accardo, A.; Aloj, L.; Aurilio, M.; Morelli, G.; Tesauro, D. Receptor binding peptides for target-selective delivery of nanoparticles encapsulated drugs, Int. J. Nanomed. 2014, 9, 1537– 1557. (11) Gao, W. W.; Chan, J. M.; Farokhzad, O. C. pH-Responsive Nanoparticles for Drug Delivery, Mol. Pharmaceutics 2010, 7, 1913-1920. (12) Klouda, L. Thermoresponsive hydrogels in biomedical applications: A seven-year update, Eur. J. Pharm. Biopharm. 2015, 97, 338-349. (13) Li, Y. P.; Xiao, K.; Zhu, W.; Deng, W. B.; Lam, K. S. Stimuli-responsive cross-linked micelles for on-demand drug delivery against cancers, Adv. Drug Deliv. Rev. 2014, 66, 58-73. (14) Zhao, X. B.; Liu, P. Reduction-Responsive Core−Shell−Corona Micelles Based on Triblock Copolymers: Novel Synthetic Strategy, Characterization, and Application As a Tumor Microenvironment-Responsive Drug Delivery System, ACS Appl. Mater. Interface 2015, 7, 166174. (15) Bui, Q. N.; Li, Y.; Jang, M.-S.; Huynh, D. P.; Lee, J. H.; Lee, D. S. Redox- and pHSensitive Polymeric Micelles Based on Poly(β-amino ester)-Grafted Disulfide Methylene Oxide Poly(ethylene glycol) for Anticancer Drug Delivery, Macromolecules 2015, 48, 4046-4054. (16) Zhu, W. P.; Wang, Y.; Cai, X.; Zha, G. Y.; Luo, Q. L.; Sun, R.; Li, X. D.; Shen, Z. Q. Reduction-triggered release of paclitaxel from in situ formed biodegradable core-cross-linked micelles, J. Mater. Chem. B 2015, 3, 3024-3031.


24

Page 25 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17) Xu, Z. G.; Liu, S. Y.; Kang, Y. J.; Wang, M. F. Glutathione-Responsive Polymeric Micelles Formed by a Biodegradable Amphiphilic Triblock Copolymer for Anticancer Drug Delivery and Controlled Release, ACS Biomater. Sci. Eng. 2015, 1, 585-592. (18) Li, D. W.; Bu, Y. Z.; Zhang, L. N.; Wang, X.; Yang, Y. Y.; Zhuang, Y. P.; Yang, F.; Shen, H.; Wu, D. C. Facile Construction of pH- and Redox-Responsive Micelles from a Biodegradable Poly(β-hydroxyl amine) for Drug Delivery, Biomacromolecules 2016, 17, 291300. (19) Bahrami, B.; Mohammadnia-Afrouzi, M.; Bakhshaei, P.; Yazdani, Y.; Ghalamfarsa, G.; Yousefi, M.; Sadreddini, S.; Jadidi-Niaragh, F.; Hojjat-Farsangi, M. Folate-conjugated nanoparticles as a potent therapeutic approach in targeted cancer therapy, Tumor Biology 2015, 36, 5727-5742. (20) Guo, M.; Que, C. L.; Wang, C. H.; Liu, x. Z.; Yan, H. S., Liu, K. L. Multifunctional superparamagnetic nanocarriers with folate-mediated and pH-responsive targeting properties for anticancer drug delivery, Biomaterials 2011, 32, 185-194. (21) Tian, K.; Zeng, J.; Zhao, X. B.; Liu, L.; Jia, X.; Liu, P. Synthesis of multi-functional nanocapsules via interfacial AGET ATRP in miniemulsion for tumor micro-environment responsive drug delivery, Colloid Surf. B-Biointerface 2015, 134, 188-195. (22) Zhen, Y.; Wan, S.; Liu, Y.; Yan, H.; Shi, R.; Wang, C. Atom transfer radical polymerization of solketal acrylate using cyclohexanone as the solvent, Macromol. Chem. Phys. 2005, 206, 607-612.


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

(23) Zhao, X. B.; Liu, P. Hydrophobic-Polymer-Grafted Graphene Oxide Nanosheets as an Easily Separable Adsorbent for the Removal of Tetrabromobisphenol A, Langmuir 2014, 30, 13699-13706. (24) Liu, C.; Thormann, E.; Claesson, P. M.; Tyrode, E. Surface Grafted Chitosan Gels. Part II. Gel Formation and Characterization, Langmuir 2014, 30, 8878-8888.

(25) Donnan, F. G. The theory of membrane equilibria, Chem. Rev. 1924, 1, 73-90. (26) Liu, L.; Liu, P. Synthesis strategies for disulfide bond-containing polymer based drug delivery system for reduction-responsive controlled release, Front. Mater. Sci. 2015, 9, 211-226. (27) Tian, Y.; Ravi, P.; Bromberg, L.; Hatton, T. A.; Tam, K. C. Synthesis and Aggregation Behavior of Pluronic F87/Poly(acrylic acid) Block Copolymer in the Presence of Doxorubicin, Langmuir 2007, 3023, 2638-2646. (28) Zeng, J.; Du, P. C.; Liu, L.; Li, J. G.; Tian, K.; Jia, X.; Zhao, X. B.; Liu, P. Superparamagnetic Reduction/pH/Temperature Multistimuli-Responsive Nanoparticles for Targeted and Controlled Antitumor Drug Delivery, Mol. Pharmaceutics 2015, 12, 4188-4199. (29) Saito, G.; Swanson, J. A.; Lee, K. D. Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities, Adv. Drug Deliv. Rev. 2003, 55, 199-215. (30) Pan, Y. J.; Chen, Y. Y.; Wang, D. R.; Wei, C.; Guo, J.; Lu, D. R.; Chu, C. C.; Wang, C. C. Redox/pH dual stimuli-responsive biodegradable nanohydrogels with varying responses to dithiothreitol and glutathione for controlled drug release, Biomaterials 2012, 33, 6570-6579.


26

Page 27 of 28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(31) Yi, X. Q.; Zhang, G.; Zhao, D.; Xu, J. Q.; Zhong, Z. L.; Zhuo, R. X.; Li, F. Preparation of pH and redox dual-sensitive core crosslinked micelles for overcoming drug resistance of DOX, Polym. Chem. 2016, DOI: 10.1039/C5PY01783A. (32) Jia, X.; Zhao, X. B.; Tian, K.; Zhou, T. T.; Li, J. G.; Zhang, R. N.; Liu, P. Fluorescent Copolymer-Based Prodrug for pH-Triggered Intracellular Release of DOX, Biomacromolecules 2015, 16, 3624-3631.


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 28

For Table of Contents Use Only

pH/reductant dual-responsive core-crosslinked micelles via facile in-situ ATRP for tumortargeted delivery of anticancer drug with enhanced anticancer efficiency Kun Tian, Xu Jia, Xubo Zhao, and Peng Liu*


28

ATRP for Tumor-Targeted - American Chemical Society

Recommend Documents