Ultrasensitive GSH-Responsive Ditelluride-Containing Poly(ether

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Ultra-Sensitive GSH-Responsive Ditelluride-Containing Poly(ether-urethane) Nanoparticles for Controlled Drug Release Yangyun Wang, Lina Zhu, Yong Wang, Liubing Li, Yufeng Lu, Liqin Shen, and Leshuai Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b14639 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016

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ACS Applied Materials & Interfaces

Ultra-Sensitive

GSH-Responsive

Ditelluride-Containing

Poly(ether-urethane) Nanoparticles for Controlled Drug Release Yangyun Wang†,‡,*,#, Lina Zhuǁ,#, Yong Wang†,‡, Liubing Liǁ, Yufeng Luǁ, Liqin Shenǁ,*, Leshuai W. Zhang†,‡,* † School for Radiological & Interdisciplinary sciences (RAD-X), and School of Radiation Medicine and Protection, Soochow University, Suzhou, 215123, Jiangsu, China. ‡ Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, 199 Renai Road, Suzhou Industrial Park, Suzhou, 215123, Jiangsu, China. ǁ The Second Affiliated Hospital of Soochow University, 1055 Sanxiang Road, Suzhou, 215004, Jiangsu, China.

1

ACS Paragon Plus Environment


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ABSTRACT A novel ultra-sensitive redox-responsive system for the controlled release of doxorubicin (DOX) was fabricated by ditelluride-containing poly(ether-urethane) copolymers. In this study, the ditelluride group was introduced for the first time into water-soluble copolymers used for drug delivery. Doxorubicin loaded in the copolymer nanoparticles can be released in a controlled manner through the cleavage of

ditelluride

bonds

by

glutathione

(GSH).

The

ditelluride-containing

poly(ether-urethane) nanoparticles were demonstrated to be biocompatible as drug delivery vehicles, therefore opening a new avenue in drug delivery systems for chemotherapy. Furthermore, the in vitro and in vivo studies revealed that the DOX-loaded ditelluride-containing poly(ether-urethane) nanoparticles exhibited efficient uptake in cancer cells, specific tumor targeting and antitumor activity, indicating their excellent potential as novel nanocarriers for drug delivery and cancer therapy.

KEYWORDS: Ditelluride-containing Poly(ether-urethane)s; Redox-responsive Nanoparticles;

Ultra-sensitive

Nanocarriers;

Controlled

Chemotherapy

2


Drug

Delivery;

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INTRODUCTION Various nanocarriers such as liposomes, vesicles and micelles are currently gaining attention for medical purposes, and are increasingly being used for drug and gene delivery.1,2 Although they are effective for temporal control of the drug release process, drug delivery systems based on biodegradable polymeric nanocarriers lack the spatial control desired for tissue specific targeting.3,4 Moreover, degradation of the nanocarriers can cause their fragmentation into a variety of sizes, which may create unexpected safety issues.5 To obtain greater control over the drug delivery to the targeted place at the expected time, smart release systems have been proposed and investigated, such as stimuli-responsive nanomaterials.6,7 Stimuli-responsive nanocarriers are capable of responding with numerous physicochemical alterations when stimulated by a minute change in the surrounding environment.8,9 To enhance the spatial and temporal control over the drug release process, a number of stimuli-sensitive nanocarriers have been constructed in the past thirty years. These nanocarriers exhibited morphology changes such as dissolution, swelling and collapse in response to internal stimulus (concentration changes of glucose or pH changes) or external stimulus (light or temperature).10,11 For example, slightly acidic environments are found in endosomes, lysosomes and cancerous tissues, as compared to the normal physiological pH of 7.4. Therefore, pH-sensitive nanocarriers have been proposed and synthesized for specific drug release in the endosomal/lysosomal compartments of the tumor site.12-15 Other than pH differences in the tumor microenvironment, the amount of reducing glutathione (GSH) available

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in the cells is 100-1000 folds higher than that in the extracellular microenvironment, which has been recently utilized for the smart design of stimuli-responsive nanocarriers. In addition, it should be emphasized that the tumor tissues are normally hypoxic, with at least four-fold higher amount of GSH compared to normal tissues.16-18 Therefore, stimuli-responsive nanocarriers are superior to other nanocarrier systems due to their self-controlled drug release at the spatial and temporal levels. Despite the remarkable advances in stimuli-responsive nanocarriers, their relatively slow and incomplete drug release before nanoparticle elimination in vivo significantly restricts the therapeutic efficacy and bioavailability of the drugs.19,20 Therefore, it is important to develop novel nanocarriers that can respond rapidly to the specific microenvironment in the cancer cells. In the past twenty years, chalcogen-containing polymers have been intensively investigated as new biomaterials that can function as nanocarriers for drug delivery, own to their unique chemical and biological properties.21,22 Zhang, Xu and their coworkers have developed a series of stimuli-responsive nanoparticles using selenium-containing block co-polymers, which are sensitive to oxidizing or reducing agents at very low concentrations.23-26 In contrast to commonly constructed sulfur- and selenium-containing polymers,27-31 scant attention has been paid to tellurium-containing copolymers. Tellurium is an important micronutrient for human health. The bigger radius and weaker electronegativity of tellurium atom compared to selenium and sulfur lead to lower bond energy of the homonuclear and heteronuclear single bond of tellurium.32 For

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example, the energy of Te-Te covalent bond is estimated to be 149 kJ/mol, which is lower than that of the S-S bond (240 kJ/mol) or Se-Se bond (192 kJ/mol), suggesting that the ditelluride-containing polymers are more sensitive to cleavage under reducing environments.33 Recently, Xu’s group developed a novel tellurium-containing polymer, which was ultra-sensitive to reactive oxygen species (ROS) and low dose of gamma radiation.34-37 However, very little attention has been paid until now to the introduction of the ditelluride group into copolymers. In this study, an efficient one-pot approach was used to produce highly sensitive redox-responsive ditelluride-containing poly(ether-urethane) nanoparticles for smart drug delivery.38-44 Ditelluride bonds were introduced into the copolymers for the first time and polymerized repeatedly between the hydrophobic and hydrophilic segments on the polymer chains. We also investigated the physicochemical and biological properties of the new ditelluride-containing nanoparticles, including: (1) the response of the ditelluride-containing poly(ether-urethane) nanoparticles to GSH stimuli, (2) kinetics of DOX release from DOX-loaded ditelluride-containing poly(ether-urethane) nanoparticles in response to GSH and (3) targeted and efficient drug delivery of DOX-loaded ditelluride-containing poly(ether-urethane) nanoparticles in vitro and in vivo. As far as we know, this is the first study on the GSH-responsive drug release based on ditelluride-containing poly(ether-urethane) nanoparticles for controlled drug delivery.

RESULTS and DISCUSSION

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Figure 1. (A) Synthesis of di-(1-hydroxylundecyl) ditelluride. (B) 1H NMR spectrum of di-(1-hydroxylundecyl) ditelluride in CDCl3. (C) 125Te NMR spectrum of di-(1-hydroxylundecyl) ditelluride.

Synthesis and Characterization. The aim of this work was to develop a redox-responsive ditelluride-containing system and to investigate its antitumor activity in vitro and in vivo. First, the ditelluride group was introduced into a diol structure through the reaction between disodium ditelluride and 11-bromoundecanol, and the resulting product possessed the desirable solubility. The synthetic scheme is shown

in

Figure

1A.

Figure

1B

shows

the

1

H

NMR

spectrum

of

di-(1-hydroxylundecyl) ditelluride. The peaks were observed at 3.64 and 2.61 ppm correspond to methylene protons connected to the hydroxyl group and ditelluride group, respectively. The relative number of peaks at 1.72-1.27 ppm was 36, which is close to the theoretical number of residual methylene protons.

127

Te-NMR of the

di-(1-hydroxylundecyl) ditellurid shows a peak at 474.7 ppm with diphenyl ditelluride as reference (δ=665 ppm) (Figure 1C). To distinguish di-(1-hydroxylundecyl) ditelluride

from

di-(1-hydroxylundecyl)

telluride,

mass

spectrum

of

the

di-(1-hydroxylundecyl) ditelluride was also conducted. Calculated molar mass (M) of

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di-(1-hydroxylundecyl) ditelluride is about 597.81 g/mol, where measured M+2Na+ is about 643.43 g/mol. All these results indicate the successful synthesis of di-(1-hydroxylundecyl) ditelluride.

Scheme 1. Synthetic route for PEG-PUTeTe-PEG copolymer.

The amphiphilic copolymer PEG-PUTeTe-PEG was then synthesized via sequential one-pot coupling reactions of poly(ethylene glycol) (PEG), hexamethylene diisocyanate (HDI) and di-(1-hydroxylundecyl) telluride (Scheme 1). The chemical structure, molecular weight (MW) and polydispersity indices (PDI) of the obtained copolymers were characterized by FT-IR (Figure S1, in the supporting information), 1

H-NMR (Figure 2) and GPC (Figure S2). In the FT-IR spectrum of

PEG-PUTeTe-PEG copolymer (Figure S1), the bands at 1720 cm-1 and 3340 cm-1 are indicative of the C=O and N-H stretching of urethane groups, respectively. The disappearance in the absorbance at ~2270 cm-1 of the copolymer revealed that isocyanate groups were reacted completely during the polymerization process (Figure S1). Figure 2 shows the 1H NMR spectrum of PEG-PUTeTe-PEG copolymer, with the peaks at 7.01-7.22 ppm corresponding to -NHCOO- group protons of the copolymer backbone. The relative integration ratio of the peak corresponding to PEG units (g,

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4.03 ppm) to the peak of di-(1-hydroxylundecyl) ditelluride units (h, 3.89 ppm) in the 1

H NMR spectra was 2.7:1, which is close to the ratio of the amount of reactants

added during the reaction, indicating that minimal reactants were left unreacted. GPC measurement was performed to determine the MW and PDI of the synthesized copolymer. Mw was determined to be 9.17×104 g/mol and the PDI was 1.22 from the typical GPC trace of PEG-PUTeTe-PEG copolymer (Figure S2). The unimodal GPC peak confirms the formation of the PEG-PUTeTe-PEG copolymer through polymerization.

Figure 2. 1H NMR spectrum of PEG-PUTeTe-PEG copolymer in DMSO-d6.

Redox Sensitivity of PEG-PUTeTe-PEG Nanoparticles. Di-(1-hydroxylundecyl) ditelluride has a sensitive ditelluride bond which can be rapidly cleaved in the presence of a reducing reagent, leading to dissociation of the nanoparticles. This process of dissociation is always rapid and can enable the complete release of encapsulated drugs. To investigate the redox sensitivity of PEG-PUTeTe-PEG nanoparticles, the size changes and size distributions of blank nanoparticles in response to 10 mM GSH were

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evaluated using dynamic light scattering (DLS) measurement at different time intervals. In the presence of GSH, there was rapid disassembly of the nanoparticles, resulting in nanoparticle agglomerates with a large size distribution within 5 min (Figure 3A). The shape and size distribution of PEG-PUTeTe-PEG nanoparticles before and after reduction were further characterized by TEM (Figure 3B and 3C). The nanoparticles disassembled and switched into an amorphous state with varying sizes in the presence of GSH, which is in accordance with the DLS measurements. The sensitive degradation of the nanoparticles is due to the cleavage of ditelluride bonds triggered by GSH.

Figure 3. (A) DLS of PEG-PUTeTe-PEG nanoparticles with or without GSH. (B) TEM images of blank PEG-PUTeTe-PEG nanoparticles without GSH. (C) TEM images of blank PEG-PUTeTe-PEG nanoparticles with GSH for 15 min.

In vitro DOX Release from DOX-Loaded Poly(ether-urethane) Nanoparticles. DOX is currently known as one of the most efficient drugs for breast cancer therapy 9



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and has been widely used as a classical small molecule for drug delivery research due to its fluorescent ability. DOX-loaded polymer nanoparticles were prepared and controlled

drug

release

was

investigated.

To

remove

DOX

residues,

PEG-PUTeTe-PEG solution was dialyzed in distilled water protected from light until minimal fluorescence of DOX was detected in the water outside the dialysis bag. Mechanistically, the drug may be loaded into the nanoparticles via hydrophobic interactions between DOX and the hydrophobic segments in the copolymer. The drug loading content was 17.3% and entrapment efficiency was 84.1%. For comparison purposes, DOX-loaded PEG-PUC6-PEG nanoparticles (denoted as C6-DOX) were prepared from PEG-PUC6-PEG copolymer and used as the negative control, assuming the non-responsiveness of DOX release from C6-DOX. The stability of encapsulated drugs is critical for tumor therapy, especially in the case of controlled release drugs. However, nanoparticles formed through hydrophobic interactions in aqueous solution are usually unstable in physiological environment.45 Therefore, the stability of the DOX-loaded nanocarriers needs to be characterized before their applications. Figure S3 shows that no significant agglomeration was observed by DLS even at day 21. During that period of time, the nanoparticle sizes remained at 205-220 nm and single factor analysis of variance shows p

Ultrasensitive GSH-Responsive Ditelluride-Containing Poly(ether

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