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A Bioreducible Poly(ethylene glycol)-Triphenylphosphonium Conjugate as a Bioactivable Mitochondria-Targeting Nano-carrier Zehedina Khatun, Yeon Su Choi, Yu Gyeong Kim, Kwonhyeok Yoon, Md Nurunnabi, Li Li, Eunji Lee, Han Chang Kang, and Kang Moo Huh Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01324 • Publication Date (Web): 03 Mar 2017 Downloaded from http://pubs.acs.org on March 4, 2017

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A Bioreducible Poly(ethylene glycol)-Triphenylphosphonium Conjugate as a Bioactivable Mitochondria-Targeting Nano-carrier

Zehedina Khatun,1, ‡ Yeon Su Choi,2, ‡ Yu Gyeong Kim,1 Kwonhyeok Yoon,1 Md Nurunnabi,1 Li Li,1 Eunji Lee,3 Han Chang Kang,2,* and Kang Moo Huh1,* 1

Department of Polymer Science and Engineering, Chungnam National University, Daejeon,

34134, Republic of Korea 2

Department of Pharmacy, Integrated Research Institute of Pharmaceutical Sciences, and BK21

PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggido 14662, Republic of Korea 3

Graduate School of Analytical Science and Technology, Chungnam National University,

Daejeon 34134, Republic of Korea

Corresponding Authors * Prof. Kang Moo Huh, Ph.D., Department of Polymer Science and Engineering, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea, Tel: +8242-821-6663, Fax: +82-42-821-8910, E-mail address: [email protected] * Prof. Han Chang Kang, Ph.D., Department of Pharmacy, Integrated Research Institute of Pharmaceutical Sciences, and BK21 PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea, Tel: +82-2-21646533, Fax: +82-2-2164-4059, E-mail address: [email protected]

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ABSTRACT Bioactivable nano-carrier systems have favorable characteristics such as high cellular uptake, target specificity, and an efficient intracellular release mechanism. In this study, we developed a bioreducible methoxy polyethylene glycol (mPEG)-triphenylphosphonium (TPP) conjugate (i.e., mPEG-(ss-TPP)2 conjugate) as a vehicle for mitochondrial drug delivery. A bioreducible linkage with two disulfide bond-containing end groups was used at one end of the hydrophilic mPEG for conjugation with lipophilic TPP molecules. The amphiphilic mPEG-(ssTPP)2 self-assembled in aqueous media, thereby forming core-shell structured nanoparticles (NPs) with good colloidal stability, and efficiently encapsulated the lipophilic anticancer drug doxorubicin (DOX). The DOX-loaded mPEG-(ss-TPP)2 NPs were characterized in terms of their physicochemical and morphological properties, drug-loading and release behaviors, in vitro anticancer effects, and mitochondria-targeting capacity. Our results suggest that bioreducible DOX-loaded mPEG-(ss-TPP)2 NPs can induce fast drug release with enhanced mitochondrial uptake and have a better therapeutic effect than non-bioreducible NPs.

KEYWORDS:

Bioactivable

nano-carrier,

PEG

conjugates,

Mitochondrial

targeting,

Triphenylphosphonium, Doxorubicin.


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INTRODUCTION Numerous strategies for nano-sized drug delivery systems have recently been investigated for the selective delivery of therapeutic agents into targeted compartments (e.g., organs, tissues, cells, or subcellular organelles), thereby avoiding off-target effects (e.g., systemic toxicity and innumerable side effects) and higher dosing requirements.1-4 However, organ-, tissue-, or celllevel passive and active targeting drug carriers are unlikely to be sufficient to elicit a maximal therapeutic response because many drug targets are localized in particular subcellular compartments (e.g., the nucleus, mitochondria, cytosol).5-7 Therefore, a drug carrier platform that can specifically target not only the tissue and the cells of interest but also the subcellular organelles is an intriguing tool that could maximize the therapeutic index of the encapsulated active ingredients in the treatment of disease.8-11 Among the subcellular organelles, mitochondria are a favorable target for delivery of therapeutic agents to treat various diseases such as cancer, autism, aging and mitochondrial dysfunctional related heart disease because they are crucial for the generation of bioenergy in eukaryotic cells as well as for the regulation of apoptosis,12, 13 cell signaling, proliferation,7, 14, 15 and drug resistance16-18. Recently, approaches for targeting the mitochondria by conjugation of mitochondriotropic molecules (e.g., XJB peptide and chemical compounds such as triphenylphosphonium [TPP]) to drugs or nano-carriers have been extensively investigated to improve mitochondrial accumulation, on the basis of their high binding affinity to the inner mitochondrial membranes.7,

15, 19-22

Specifically, TPP facilitates the binding and transport of

drugs or nano-carriers across the highly negatively charged mitochondrial membrane because it is sufficiently lipophilic and has a positive charge.23 Nevertheless, selective targeting of drugs to the mitochondria remains a challenge because other essential natural barriers impede the effects


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of many therapeutic molecules. The exposed TPP molecules in TPP-linked drug carriers or drugs may negatively affect either the targeting efficiency or the therapeutic activity. For instance, because TPP molecules produce a positive zeta-potential on the surface of the nanoparticles (NPs) they occupy, the NPs may non-specifically interact with blood components, thus leading to serum inhibition, rapid clearance from the blood circulation, and opsonization.24-26 In addition, although TPP-linked NPs can aid in cellular internalization via electrostatic attraction between the positive charges of TPP-linked NPs and the negative charges of the plasma membrane, the cationic character of TPP induces non-specific cytotoxicity via destabilization of the plasma membrane.20 In this study, to pursue a solution for these issues by using TPP as a mitochondria-targeting ligand, we designed disulfide-linked poly(ethylene glycol)-(TPP)2 (PEG-(ss-TPP)2) conjugates and their self-assembled NPs (Figure 1). The amphiphilic PEG-(ss-TPP)2 conjugates selfassemble in aqueous media, thus forming bioreducible NPs composed of a hydrophilic PEG shell and a hydrophobic TPP core. On the basis of the constituent components (i.e., PEG, TPP, and disulfide bonds), the unique biomimetic architecture (i.e., a phospholipid-like structure), and the bioactivatable morphological change (due to the efficient breakage of disulfide bonds after cellular uptake), the PEG-(ss-TPP)2 conjugates and their self-assembled NPs may be an efficient mitochondrial targeting nano-carrier system for the treatment of various diseases, particularly cancer. First, a hydrophilic PEG shell may endow the NPs with a “stealth” effect, thereby allowing them to avoid clearance from the serum and improving the circulation time. Longcirculating NPs may show enhanced selective accumulation in organs/tissues with a fenestrated or sinusoidal capillary (particularly in solid tumors), on the basis of the well-known EPR effect. Additionally, the cellular uptake of the PEGylated NPs is expected to occur via endocytosis, thus


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bypassing the efflux pump resistance. Second, the lipophilic and cationic TPP molecules in PEG(ss-TPP)2 conjugates were designed to play three roles: their essential roles as a hydrophobic component for constructing self-assembled NPs and encapsulating hydrophobic payloads and another role as a guide chemical for targeting the mitochondria. In addition, the biomimetic architecture used to introduce two TPP molecules to one end of a single PEG chain, thus enhancing the hydrophobicity of the conjugates, may facilitate their self-assembling properties and provide a high drug loading capacity. Importantly, the cancer-killing drug doxorubicin (DOX) was efficiently loaded into the NP via hydrophobic interactions and/or π-π stacking between TPP and the DOX molecules. Third, after internalization of the drug-loaded PEG-(ssTPP)2 NPs into the target cells, the disulfide linkages between PEG and TPP can be cleaved in response to the intracellular redox potential (e.g., glutathione), thus resulting in the shedding of the PEG shells from the NPs, exposing the TPP moieties surrounding the NPs and then triggering TPP-driven mitochondrial accumulation of the therapeutic agent. Our DOX-loaded PEG-(ssTPP)2 NPs (i.e., PEG-(ss-TPP)2/DOX NPs) based on bioreducible PEG shedding and TPP exposure mechanisms caused increased drug uptake into the mitochondria, consequently eliciting a maximum therapeutic response of the encapsulated DOX. In this study, the PEG-(ss-TPP)2 conjugates and their DOX-loaded NPs were characterized in terms of their physicochemical and morphological properties, drug-loading and release behavior, in vitro anticancer effects, and mitochondria-targeting capacity.

MATERIALS AND METHODS Materials. dihydrochloride,

Methoxy

polyethylene

p-nitrophenyl

glycol

chloroformate

(mPEG, (pNC),

MW

2000

Da),

cystamine

N-(3-(dimethylamino)propyl)-N′-


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ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), triethylamine (TEA), acetonitrile, dimethyl sulfoxide (DMSO), dichloromethane (DCM), 9,10-dimethylanthracene (DMA), triphenylphosphine, 6-bromohexanoic acid, dithiothreitol (DTT), bovine serum albumin (BSA), phosphate buffered saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI1640 medium, sodium bicarbonate, 4-(2-hydroxy-ethyl)-1-piperazine (HEPES), D-glucose, penicillin-streptomycin antibiotics, fetal bovine serum (FBS), trypsin-EDTA, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Hoechst33342, and a Nuclei PURE Prep kit were purchased from Sigma-Aldrich Company (St. Louis, MO, USA). 2-Amino1,3-propanediol (serinol) and Spectra/Por membranes were obtained from Tokyo Chemical Industry (Tokyo, Japan) and Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA), respectively. Doxorubicin hydrochloride (DOX·HCl) and a mitochondria isolation kit were from MedKoo Biosciences (Chapel Hill, NC, USA) and BioVision (Milpitas, CA, USA), respectively. MitoTracker® Deep Red FM was purchased from Life Technologies (Grand Island, NY, USA).

Synthesis of a bioreducible mPEG derivative with two amino end groups (mPEG-(ssNH2)2). Cystamine-derivatized mPEG, mPEG-(ss-NH2)2, was synthesized via a multi-step route according to the methodology in a previous report, with minor modifications.27 In brief, mPEG (5 mmol) was dissolved in acetonitrile (20 mL) and then reacted with pNC (62.5 mmol) in the presence of TEA (62.5 mmol) at room temperature (RT) under inert N2 gas for 24 h. The crude product was filtered, precipitated in diethyl ether, and dried in vacuo. Then, pNC-activated mPEG (3.39 mmol) was reacted with serinol (31.41 mmol) in DMSO at RT under inert N2 gas for 36 h, and this was followed by dialysis against deionized water (DIW) for 2 days, using a dialysis membrane (MWCO 1000 Da), and lyophilization. The hydroxyl end groups of the serinol-derivatized mPEG (mPEG-serinol) were again activated by pNC via the aforementioned


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methods. To obtain bioreducible mPEG-(ss-NH2)2, the pNC-activated mPEG-serinol (2 g) was reacted with cystamine dihydrochloride (3.68 g) in DMSO in the presence of TEA (1.65 mL) at RT for 24 h. The crude product was purified by filtration, precipitated in ether, and washed with DCM. mPEG-(ss-NH2)2 was obtained as a white powder after vacuum drying. To prepare a nonbioreducible mPEG-(ethylenediamine)2 (i.e., mPEG-(NH2)2) for a non-bioreducible PEG-(TPP)2 conjugate as a control, the pNC-activated mPEG-serinol was also reacted with ethylenediamine (59 mmol) to form a non-bioreducible mPEG-(NH2)2 by using the same conditions and purification methods. The synthesis of mPEG-(ss-NH2)2 and mPEG-(NH2)2 was confirmed via 1

H NMR (600 MHz, CDCl3) (Bruker, MA, USA).

Synthesis and characterization of the bioreducible mPEG-(ss-TPP)2 conjugate. Triphenylphosphine (5 mmol) and 6-bromohexanoic acid (6 mmol) were dissolved in dry acetonitrile and reacted at 80 oC under inert N2 gas for 16 h. After cooling of the reaction solution to RT, a white precipitate was obtained in a crystallized form. The formation of triphenylphosphonium hexanoic acid (TPP-COOH) was confirmed by 1H-NMR (600 MHz, DMSO-d6), and the purity was confirmed by HPLC. For the synthesis of mPEG-(ss-TPP)2 conjugate, TPP-COOH (6 mmol) was dissolved in DMSO and activated with EDC (6 mmol) and NHS (6 mmol). After a 30 min reaction, mPEG(ss-NH2)2 (1 mmol) was added, and the reaction was allowed to proceed at RT for 12 h. The resultant mixture was dialyzed against DIW for 1 day and then lyophilized. As a control, the non-bioreducible mPEG-(TPP)2 conjugate was synthesized using the same protocol. The chemical structures of mPEG-(ss-TPP)2 and mPEG-(TPP)2 conjugates were confirmed by 1HNMR (600 MHz, DMSO-d6) and FT-IR (Nicolet 380, Thermo Scientific, MA, USA; KBr), and their molecular weights were determined using an Agilent 1100 gel permeation chromatography


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(GPC) system (Agilent Technologies, CA, U.S.A.). The GPC settings and methods were as described in our previous report.28 The GPC traces of mPEG-TPP2 and mPEG-(ss-TPP)2 conjugate were recorded using by a Agilent 1100 gel permeation chromatography system equipped with quaternary pump and refractive index detector using Agilent PL gel 5 µm mixedC and mixed-D (300 mm×7.5 mm) columns. The eluent was THF, and the flow rate was 1.0 mL/min. The temperature of columns was set at 40 °C and polystyrene standards were used to compare the relative molecular weights of the polymer conjugates.

The critical aggregation concentration of mPEG-(ss-TPP)2. The critical aggregation concentration of mPEG-(ss-TPP)2 conjugate was estimated using pyrene as a fluorescence probe.29 The concentration of the conjugate was varied from 0.0001 to 0.1 mg/mL. The concentration of pyrene in the polymer solution was fixed at 6×10-7 M. The polymer solutions with pyrene were equilibrated in the dark for 1 day at RT. The fluorescence spectra were monitored with a fluorescence spectrophotometer (Cary Eclipse, Varian, USA). The slit widths for both excitation and emission were 5 nm. The pyrene fluorescence emission was recorded at λex = 336 nm. The intensity ratio of the peaks at 374 nm to the peaks at 394 nm was plotted against the concentration of the polymers on a logarithmic scale to determine the critical aggregation concentration. The critical aggregation concentration of mPEG-(TPP)2 conjugate was evaluated in a similar fashion.

Encapsulation of doxorubicin (DOX) in mPEG-(ss-TPP)2 NPs. DOX·HCl (2.5 mg) was dissolved in DMSO (2.5 mL), and an equivalent amount of triethylamine (TEA) was added to the solution with stirring. The solution was stirred for 4 h and incubated overnight at RT. mPEG(ss-TPP)2 conjugate (47.5 mg) was dissolved in DMSO (5.5 mL), and a pre-prepared stock


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solution of DOX was added dropwise with vigorous stirring at RT. The solution was stirred overnight at RT in the dark, and this was followed by dialysis against deionized water for 10-12 h to exchange the DMSO solvent with an aqueous solution and to remove free DOX. After dialysis, the solution of DOX-loaded mPEG-(ss-TPP)2 NPs (i.e., mPEG-(ss-TPP)2/DOX NPs) was filtered and lyophilized. DOX-loaded mPEG-(TPP)2 NPs (i.e., mPEG-(TPP)2/DOX NPs) were prepared in a similar fashion. The amount of DOX loaded in the drug-loaded NPs was calculated via UV absorbance spectroscopy (UV-2600, Shimadzu, Japan). The mPEG-(TPP)2/DOX NPs and mPEG-(ssTPP)2/DOX NPs (1 mg/mL) were dissolved in PBS, and then the absorbance of DOX was monitored at 465 nm. The concentration of DOX was determined according to a standard curve prepared using free DOX in DMSO. The loading efficiency (LE) and loading content (LC) of DOX in the DOX-loaded NPs were calculated through the following formulas: LE (%) =

× 100,

LC (%) = × 100.

Size distribution and colloidal stability of mPEG-(ss-TPP)2/DOX NPs. The hydrodynamic size and size distribution of the DOX-loaded NPs were determined by dynamic light scattering (DLS) (ELS-Z series, Otsuka Electronics, Japan). To assess the colloidal stability of the DOX-loaded NPs, mPEG-(ss-TPP)2/DOX NPs (1 mg/mL) were dispersed in PBS containing 10% BSA and incubated in a shaking bath at 37 °C. At different times, the size of the NPs was measured by DLS. Similarly, the hydrodynamic size and size distribution of mPEG(TPP)2/DOX NPs were monitored. The reduction-triggered PEG shedding properties of mPEG(ss-TPP)2/DOX NPs were tested by dispersing mPEG-(ss-TPP)2/DOX NPs in PBS containing


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DTT (10 mM). The NP solution was incubated in a shaking bath at 37 °C, and the size change was monitored at different times. Similarly, using mPEG-(TPP)2/DOX NPs as a non-reducible counterpart, DTT-induced changes in their hydrodynamic size and size distribution were measured.

DOX release from mPEG-(ss-TPP)2/DOX NPs. A drug release study from the NPs was conducted according to our previously described method.30 The release behavior of DOX was observed in PBS and 10 mM DTT-containing solutions. Briefly, mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs (1 mg/mL) were dispersed in DTT-free PBS and DTT (10 mM)containing PBS, respectively, and then the NP solutions were placed in a dialysis membrane (MWCO 3500 Da). The dialysis bag was immersed into the same buffer solution (25 mL) and kept at a constant temperature (37 ◦C) and stirred at 100 RPM. At different times, 3 mL of the solutions was removed for analysis and an equal volume of solution was added to maintain the solution volume. The DOX release profile was determined by measuring the UV absorbance of DOX at 485 nm.

Cells and cell culture. Two cancer cell lines, HepG2 (a human hepatoma cell line) and HeLa cells (a human cervical adenocarcinoma cell line), were used to evaluate the in vitro cytotoxicity and intracellular distribution of the DOX-loaded NPs. HepG2 and HeLa cells were cultured in DMEM and RPMI1640, respectively, supplemented with 10% FBS, 1% antibiotics, and D-glucose (4.5 g/L for DMEM and 2 g/L for RPMI1640) under a humidified atmosphere of 95% air and 5% CO2 at 37 oC.


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In vitro cytotoxicity of drug-free NPs and in vitro drug effects of drug-loaded NPs. After the cells were exposed to the drug-free and drug-loaded NPs for 48 h, the change in their viability was monitored with MTT assays.31, 32 In brief, the cells (5000 cells in 0.1 mL) were seeded into a 96-well plate and incubated for 24 h. After preparation of samples (e.g., drug-free NPs, drug-loaded NPs, and the model drug) with various concentrations of either polymers or the model drug (i.e., DOX), the cells were treated with the samples for 48 h. An MTT solution (10 µL, 5 mg/mL) was added to the cells, and the cells were incubated for an additional 4 h. The MTT-containing culture medium was replaced with DMSO (0.1 mL) to dissolve the formazan crystals produced by the live cells. The absorbance (ABS) was measured at 570 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA), and the following equations were used to calculate the viability. Cell viability "%$ =

ABS)* + ,

- − ABS /) × 100 ABS+ ,

- − ABS /)

Cellular uptake (CU), nuclear uptake (NU), mitochondrial uptake (MU), and intracellular distribution of drug-loaded NPs. To evaluate the intracellular distribution of DOX-loaded NPs, their CU, NU, and MU were monitored and compared with those of DOX.20 In brief, HepG2 cells (5×105 cells in 2 mL) were seeded in 6-well plates and incubated for 24 h. After treatment with either free DOX or DOX-loaded NPs ([DOX] = 3 µg/mL) for 4 h, the uptake into the cells and the intracellular compartments (i.e., the nuclei and the mitochondria) was determined. First, to evaluate CUs of free DOX or DOX-loaded NPs, the cells were rinsed twice with DPBS and detached by trypsin-EDTA. The cellular fluorescence of DOX in cells that had taken up free DOX or DOX-loaded NPs was determined with a FACSCantoTM II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA) with a primary argon laser (532 nm)


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and a fluorescence detector (578 ± 15 nm). After 1×104 cells had been detected, the cellular DOX fluorescence of the cells located in a gated region for live cells was determined. Second, to determine the NU and MU, the nuclei and mitochondria of free DOX or DOX-loaded NPs in HepG2 cells at 4 h post-treatment were isolated, per the manufacturer’s protocols with a Nuclei PURE Prep kit or Mitochondrial Fractionation kit, respectively. The subcellular fluorescence of DOX in the subcellular organelles (i.e., the nuclei and the mitochondria) of cells that had taken up free DOX or DOX-loaded NPs was monitored in a similar manner as the measurement of their cellular fluorescence. Normalized values were used to compare the CU, NU, and MU of the DOX-loaded NPs with those of free DOX. In addition, the nuclear localization preference (NLP), the mitochondria localization preference (MLP), and the mitochondria-to-nucleus preference (MNP) of the DOX-loaded NPs were estimated. Normalized CU of DOX-loaded NPs =

CU of DOX-loaded NPs CU of free DOX

Normalized NU of DOX-loaded NPs =

NU of DOX-loaded NPs NU of free DOX

Normalized MU of DOX-loaded NPs =

MU of DOX-loaded NPs MU of free DOX

NLP of DOX-loaded NPs=

Normalized NU of DOX-loaded NPs Normalized CU of DOX-loaded NPs

MLP of DOX-loaded NPs=

Normalized MU of DOX-loaded NPs Normalized CU of DOX-loaded NPs

MNP of DOX-loaded NPs=

Normalized MU of DOX-loaded NPs Normalized NU of DOX-loaded NPs

To identify the subcellular localization and intracellular intensity of the DOX delivery with or without a carrier (e.g., NPs),20 HepG2 cells (3 × 104 cells in 0.3 mL) were seeded in a confocal


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dish and incubated for 24 h. The cells were then incubated in the presence of DOX-loaded NPs or free DOX with [DOX] = 1 µg/mL for 4 h. MitoTracker Deep Red FM (150 nM) and Hoechst 33342 (5 µg/mL) were added to stain the mitochondria at 30 min and the nuclei at 10 min, respectively, before the end of the 4 h treatment. After the cells were rinsed twice with DPBS, the intracellular distribution and fluorescence of DOX in free DOX-treated or DOX-loaded NPtreated HepG2 cells were monitored with a laser scanning confocal microscope equipped with excitation lasers (405 nm for diode, 543 nm for HeNe, and 633 nm for HeNe) and variable bandpass emission filters (LSM710; Carl Zeiss, Oberkochen, Germany).

Statistical analysis. All of the data are expressed as the mean ± standard error (SE). Data were statistically analyzed using Origin pro 8.0 software.

RESULTS AND DISCUSSION Synthesis and characterization of mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates. For the syntheses of the bioreducibile mPEG-(ss-TPP)2 conjugate and the non-bioreducible mPEG(TPP)2 conjugate, carboxylated TPP (TPP-COOH) was chemically linked with the bioreducibile mPEG-(ss-NH2)2 polymer and the non-bioreducible mPEG-(ethylenediamine)2 (i.e., mPEG(NH2)2 or mPEG-(EDA)2) polymer via a multi-step synthetic route (Figure 2(A)). First, mPEGserinol and sequentially the mPEG-(pNC)2, mPEG-(NH2)2 and mPEG-(ss-NH2)2 polymers were successfully synthesized from mPEG-pNC, as confirmed by 1H-NMR spectra (Figure 2(B)). The major peaks of mPEG-(NH2)2 polymers in the 1H-NMR spectra were assigned as follows: δ 2.8 (2H, α-CH2-), δ 3.2 (2H, β-CH2-), δ 3.4 (3 H, CH3-), and δ 3.5-3.7 (196 H, -CH2CH2-O-). The presence of mPEG and cystamine in mPEG-(ss-NH2)2 polymers was assigned by the following


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H NMR peaks: (400 MHz, CDCl3): δ = 2.6 (2 H, α-CH2-), 2.8 (2 H, β-CH2-), 3.4 (3 H, CH3-),

3.5–3.7 (196 H, -CH2-CH2-O−), and 5.3 (1 H, -NH-). Second, the bioreducible mPEG-(ss-TPP)2 conjugates and non-bioreducible mPEG-(TPP)2 conjugates were synthesized by conjugating TPP-COOH with the amino end groups of the mPEG-(ss-NH2)2 or mPEG-(NH2)2 polymers, respectively. In the final conjugates, the peaks at δ 7.6–7.9 indicated the presence of TPP (Figure 2(B)). In addition, the presence of both TPP and mPEG in the conjugates was confirmed from the FT-IR spectra of mPEG, TPP-COOH, the mPEG-(TPP)2 conjugates and mPEG-(ss-TPP)2 conjugates (Figure 2(C)). Specifically, the C–H stretching of TPP moieties was absorbed at approximately 2958 cm-1, 2923 cm-1, and 2856 cm-1, and the energy bands between 1217 and 910 cm-1 were identified as stretching of the C–P bonds and C–N bonds in TPP moieties. The peaks near 1510-1730 cm-1 indicated the presence of absorption peaks of C=O and –COO– of TPP-COOH. The presence of amide bonds in mPEG-(TPP)2 conjugates and mPEG-(ss-TPP)2 conjugates was confirmed by the absorption peaks at 1742 and 1580 cm-1. The number of TPP molecules conjugated to one PEG end was calculated on the basis of the peak integration of 1H-NMR spectra as 1.52 and 1.41 for mPEG-(TPP)2 conjugates and mPEG(ss-TPP)2 conjugates, respectively. Such low conjugation efficiency might have resulted from steric hindrance caused by the bulky chemical structure of TPP molecules. The number-average molecular weight (Mn) of the conjugates was measured by 1H-NMR and gel permeation chromatography (GPC). From the peak integration of the 1H-NMR spectra, the Mn values were estimated as 2955-2965 Da for the mPEG-(TPP)2 conjugates and 2845-2850 Da for the mPEG(ss-TPP)2 conjugates. The GPC-based Mn values for the mPEG-(EDA)2 polymers and mPEG(ss-NH2)2 polymers were 3064-3070 Da and 3104-3112 Da, respectively, and their TPP conjugation increased the Mn values to 3657-3662 Da for the mPEG-(TPP)2 conjugates and


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3720-3726 Da for the mPEG-(ss-TPP)2 conjugates. The GPC results also confirmed the successful chemical conjugation of TPP to the mPEG-(EDA)2 polymers and mPEG-(ss-NH2)2 polymers. The self-assembled NP formation of mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates was demonstrated by measuring their critical aggregation concentration at pH 7.4. From a plot of the I374/I394 ratio versus the logarithm of the polymer concentration, the critical aggregation concentration of mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates were determined to be 0.0177 mg/mL and 0.0126 mg/mL, respectively. The characteristics of the synthesized conjugates are summarized in Table 1.

Physicochemical and morphological characterization of mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs. The hydrophobic anticancer drug DOX was physically loaded into the self-assembled NPs constructed by either the non-bioreducible or bioreducible conjugates. The loading content (LC, wt%) and loading efficiency (LE, wt%) of DOX in the NPs were calculated from a DOX standard curve of UV-visible absorbance. The calculated LE and LC values of DOX were 74% and 4.6 wt%, respectively, for mPEG-(TPP)2/DOX NPs and 76% and 4.5 wt%, respectively, for mPEG-(ss-TPP)2/DOX NPs. Both conjugates efficiently encapsulated the hydrophobic drug during the self-assembly of the nanoparticles and showed LE and LC values comparable to those of typical polymer micelles, possibly because of strong hydrophobic interactions and π-π stacking between DOX and the TPP moieties. The size distribution, shape, and morphology of the mPEG-(TPP)2/DOX NPs and mPEG(ss-TPP)2/DOX NPs were characterized by scanning electron microscopy (SEM) and dynamic light scattering (DLS). The average particle diameters of the mPEG-(TPP)2/DOX NPs and


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mPEG-(ss-TPP)2/DOX NPs were 169 and 161 nm, and polydispersity index (PDI) are 0.12 and 0.26, respectively (Figure 3(A) and 3(B)). The NPs of mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates without DOX were observed to have larger sizes (317 nm and 177 nm, respectively, Figure S1). For both NPs, particle sizes of the NPs were decreased after loading the DOX. Such reduced sizes of the NPs with drug loading may result from enhancement of hydrophobic interaction between the drug molecules and the polymer chains that is a main driving force for self-assembled NP formation. The SEM images showed that all the NPs were spherical in shape, and uniformly distributed without aggregation (Figure 3(C) and 3(D)). Considering their large hydrodynamic sizes compared to those of typical polymer micelles, both NPs are considered to exist as multi-micellar aggregates with PEG shells and DOX cores.

Colloidal stability of the NPs. A long circulation time and colloidal stability of NPs in the blood stream are critical issues for systemic delivery systems. Protein adsorption on the surface of NPs may cause colloidal instability, thereby resulting in failed drug delivery by the carrier. Thus, to evaluate the colloidal stability of the mPEG-(TPP)2/DOX NPs and mPEG-(ssTPP)2/DOX NPs, the lyophilized NPs were re-dispersed into either BSA-free PBS (pH 7.4) or 10% BSA-supplemented PBS, and their size changes were monitored by DLS. BSA was selected for the study because albumin is an abundant serum protein. First, no significant size changes of the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were detected during a short-term incubation of the NPs in BSA-free PBS, and their sizes remained approximately constant for 1 day (Figure 3(E)). As time passed, a slight increase in the NP size was detected, and the size increments of both mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs at 7 days were approximately 5%, compared with the sizes of the DOX-loaded NPs at 0 days.


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Second, addition of the DOX-loaded NPs into PBS with 10% BSA resulted in a size increase of approximately 20%. The size of the DOX-loaded NPs continuously increased, reaching 232236 nm in diameter (i.e., almost 1.4-fold larger than the DOX-loaded NPs in PBS) at 7 days (Figure 3(E)). Although the size increments of both DOX-loaded NPs in 10% BSAsupplemented PBS were slightly larger than those in BSA-free PBS, the results indicated that mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs had excellent colloidal stability in aqueous media regardless of the presence of BSA, thus further suggesting that the PEGylated NPs may be used in the blood without posing a significant concern regarding serum protein absorption.

Thiol-induced transformation of mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs in the presence of DTT. The cytosolic, mitochondrial, and nuclear concentrations of reduced glutathione (GSH), a thiol chemical in the body, are 1-11 mM, and the intra-cellular concentrations of GHS are 50- to 1000-fold higher than the extracellular concentrations.2 In these thiol-rich intracellular environments, the disulfide bond-incorporating NPs may accompany significant structural disintegration or transformation, modulating the release of their payloads. To estimate the GSH-induced transformation of the non-bioreducible mPEG-(TPP)2/DOX NPs and bioreducible mPEG-(ss-TPP)2/DOX NPs in the cytosol, the lyophilized NPs were redispersed into the model thiol (10 mM)-containing PBS. DTT was used as a thiol reducing agent instead of GSH. Specifically, the thiol-induced nano-structural transformation of the designed NPs was estimated by monitoring their time-dependent changes in size distribution because the breakdown of the disulfide linkages in the NPs affected the size of the NPs in the presence of DTT (10 mM) at 37 °C.


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As shown in Figure 4(A), the time-dependent size distribution of non-bioreducible mPEG(TPP)2/DOX NPs in DTT-free PBS was nearly constant for 12 h. In DTT (10 mM)supplemented PBS, although the non-bioreducible NPs were slightly larger, the NPs did not have a markedly different time-dependent size distribution for 12 h (Figure 4(B)). The size distribution of the bioreducible mPEG-(ss-TPP)2/DOX NPs in DTT-free PBS did not significantly change over time in a similar fashion as their non-bioreducible counterpart (Figure 4(C)). However, DTT (10 mM) caused marked changes in the time-dependent size distribution of bioreducible NPs (Figure 4(D)). Specifically, the bioreducible mPEG-(ss-TPP)2/DOX NPs had a mean diameter of 161 nm and a unimodal size distribution before the NPs were exposed to DTT (10 mM)-containing PBS at 37 °C. However, for the first 30 min in the presence of DTT (10 mM), the size distribution was broadened, and the average size increased to a diameter of 294 nm. The size of the NPs continuously increased after only 1 h of incubation, and their size distribution became multi-modal, indicating that significant aggregation between the NPs occurred due to breakage of the disulfide bonds. The particle size could not be detected any more after 6 h of incubation. The time-dependent size distribution behavior in the absence or presence of DTT (10 mM) indicated that the non-bioreducible NPs did not undergo a thiol (10 mM)induced nano-structural transformation, and hence no change in their colloidal stability in the blood stream as well as the intracellular environment was evident. The results also indicated that the disulfide bonds in the bioreducible NPs rapidly dissociated in the presence of DTT to cause the destabilization of the NPs in a short time. As a result, the bioreducible NPs with excellent colloidal stability may be expected to change with the bioreductive PEG shedding mechanism into a hydrophobic TPP/DOX nano-suspension in the intracellular environment.


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Drug release profiles of the NPs. The release profiles of DOX from the non-bioreducible mPEG-(TPP)2/DOX NPs and bioreducible mPEG-(ss-TPP)2/DOX NPs were investigated in the presence or absence of DTT (10 mM) to estimate the thiol-triggered drug release patterns of both types of NPs in the intracellular environment. As shown in Figure 5, the release profiles of DOX from non-bioreducible mPEG-(TPP)2/DOX NPs and bioreducible mPEG-(ss-TPP)2/DOX NPs in the absence of DTT were not different. Additionally, the non-bioreducible NPs did not show any significant changes in their release kinetics regardless of the presence of DTT. However, the bioreducible NPs released DOX more rapidly than the non-bioreducible NPs in the presence of DTT (10 mM). The DOX release rates from bioreducible NPs were approximately 1.3-fold, 1.6fold, 1.4-fold, and 1.4-fold faster those of the non-bioreducible NPs at 1 h, 2 h, 3 h, and 6 h, respectively. Specifically, the cumulative amount of DOX released from the bioreducible NPs was approximately 75%, 88%, and 96% at 3 h, 6 h, and 12 h, respectively, whereas the amount of DOX released from the non-bioreducible NPs was approximately 55%, 65%, and 74% at 3 h, 6 h, and 12 h, respectively. The unreleased drug remaining after 12 h is considered to exist tagged with the PEG chains or inside the NPs. The burst release of DOX from both bioreducible and non-bioreducible NPs occurred in 6 h, which is a typical profile of drug release from PEGlytaed nanoparticle. However, the significant differences for bioreducible NPs were observed after 6 h with 60% of DOX release. However, almost 96% of total DOX was released in 12 h of observation. No additional release from the bioreducible NPs was detected 12 h after DTT exposure. These results indicate that the disulfide linkages of mPEG-(ss-TPP)2/DOX NPs can be reduced and broken in the thiol-rich intracellular environment, thus leading to the exposure of the nano-suspension (i.e., TPP-exposed DOX core), the destabilization of the


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hydrophobic drug core, the accelerated solubilization of hydrophobic DOX, and rapid DOX release.

In vitro cytotoxicity of mPEG-(TPP)2 and mPEG-(ss-TPP)2 NPs. In general, the materials for designing nano-sized drug delivery carriers must have good biocompatibility and negligible cytotoxicity. Although PEG has excellent biocompatibility in terms of toxicity, immunogenicity, antigenicity, and tumorigenesis.33, 34 The lipophilic cationic TPP moieties used for mitochondrial targeting may cause some cellular and mitochondrial damage, and its application as an anticancer drug has also been reported.35 In our previous study, we have found that the NPs composed of TPP-b-poly(ε-caprolactone)-b-TPP (TPCL) copolymers have significant cytotoxic effects (IC50 values of TPCL NPs ≈ 10-60 µg/mL at 48 h of treatment in HepG2 and HeLa cells), owing to the presence of cationic TPP molecules on the surface of the NPs.20 Thus, the in vitro cytotoxicity of mPEG-(TPP)2 NPs and mPEG-(ss-TPP)2 NPs was monitored to determine whether the location of TPP in the NP significantly affects cell death through cellular and mitochondrial damage. When cells were exposed to mPEG-(TPP)2 NPs and mPEG-(ss-TPP)2 NPs with ≤ 1 mg/mL of polymers for 48 h, greater than 90% of HepG2 cells (Figure 6(A)) and greater than 80% of HeLa cells (Figure 6(B)) survived. Although the viability of the mPEG-(ss-TPP)2 NP-treated cells was slightly less than that of the mPEG-(TPP)2 NP-treated cells, the viability differences were not statistically significant. The negligible cytotoxicity of mPEG-(TPP)2 NPs and mPEG(ss-TPP)2 NPs at 1 mg/mL or lower concentration allows for their application as nano-sized drug carriers. Moreover, the TPP molecules in the PEGylated NPs did not reduce cell viability, unlike TPP molecules on the surface of NPs.


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In vitro DOX effects of mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs. As drug carriers, the mPEG-(TPP)2 NPs and mPEG-(ss-TPP)2 NPs had negligible cytotoxicity and a reasonable encapsulation capacity for the hydrophobic DOX. To determine whether the NPs efficiently delivered therapeutic agents to cells, the delivery efficiencies for DOX of the mPEG(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were evaluated and compared with free DOX in an MTT-based cell viability assay (Figure 7). Although lower DOX effects of PEGylated DOX NPs would be possible, owing to the slow internalization of typical PEGylated carriers, interestingly, both the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs had good DOX delivery efficiencies. At 48 h post-treatment, the IC50 values for free DOX in HepG2 (Figure 7(A)) and HeLa (Figure 7(B)) cells was approximately 3.8 µg/mL and 1.6 µg/mL, respectively. In HepG2 cells, mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were approximately 1.9-fold and 3.8-fold, respectively, when compared to free DOX. In HeLa cells, the IC50 value of mPEG-(TPP)2/DOX NPs was similar to that of free DOX, whereas mPEG-(ss-TPP)2/DOX NPs still showed a 3.2-fold greater killing activity than that of free DOX. Interestingly, the DOX effects of mPEG-(ss-TPP)2/DOX NPs were approximately 2- to 3-fold greater than those of mPEG-(TPP)2/DOX NPs. The various DOX effects of PEGylated TPP-based NPs may have been affected by the presence of cytosol-specific biodegradable bonds in the NPs, because the disulfide bonds in mPEG-(ss-TPP)2/DOX NPs would be broken in the thiol-rich cytosol, thus resulting in the formation of a lipocationic TPP-exposed DOX NP, unlike mPEG-(TPP)2/DOX NPs. The results indicate that mPEG-(ss-TPP)2 NPs may be a potential drug carrier for cytosolic delivery of therapeutic agents.


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Cellular uptake and intracellular distribution of DOX delivered with mPEG(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs. Although mPEG-(ss-TPP)2/DOX NPs were PEGylated, they had better DOX effects. To understand how the NPs designed with both mitochondria-targeting TPP molecules and cytosol-specific biodegradable disulfide bonds improved the therapeutic effects of the delivered DOX, the cellular uptake and intracellular distribution of the DOX delivered by mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were determined. After treatment of HepG2 cells with the DOX-loaded NPs or free DOX for 4 h, their cellular uptake (CU), nuclear uptake (NU), and mitochondrial uptake (MU) were estimated from the cellular, nuclear, and mitochondrial DOX fluorescence monitored by flow cytometry. In comparison, the CU, NU, and MU of the DOX-loaded NPs were normalized to those of the free DOX (Figure 8(A)). First, the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs had an approximately 1.49-fold and 1.32-fold lower intracellular fluorescence intensity (i.e., CU), respectively, than that of free DOX, thus indicating that the cellular drug-delivery efficiency of the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs was approximately 67% and 76%, respectively, of the cellular drug-delivery efficiency of the free DOX. This phenomenon may have been caused by differences in the internalization mechanism, because hydrophobic DOX is generally taken up through adsorptive endocytosis, passive diffusion through the plasma membrane, or both, but PEGylated NPs are typically taken up through slower fluid-phase endocytosis. Second, although more drugs could be delivered into cells, the drugs must reach the subcellular compartments to exert therapeutic activity. However, the fluorescence intensity of DOX in DOX-intercalated DNA is approximately 10-fold lower than that of free DOX,36 thus indicating that DOX fluorescence in the mitochondria and the nucleus may be approximately 10-


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fold lower than that of the cytosolic DOX, although the same amount of DOX may be present in each of these three compartments. Therefore, we isolated the nuclei and the mitochondria for further analysis of the subcellular DOX fluorescence intensity in mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs because the therapeutic action of DOX must be exerted in the nucleus and the mitochondria.20 Compared with the subcellular uptake of free DOX, mPEG(TPP)2/DOX NPs had an approximately 1.69-fold lower NU and a 1.25-fold higher MU, whereas the NU and MU of mPEG-(ss-TPP)2/DOX NPs were approximately 2.56-fold lower and 1.74fold higher, respectively. The results can be stated more simply: the non-bioreducible mPEG(TPP)2/DOX NPs and bioreducible mPEG-(ss-TPP)2/DOX NPs, compared with free DOX, had lower CUs and NUs but higher MUs. Further quantitative analyses of the intracellular DOX distribution were carried out by determining three new parameters, that is, the nuclear localization preference (NLP), the mitochondrial localization preference (MLP), and the mitochondria-to-nucleus preference (MNP) (Figure 8(B)). Our previous studies have reported that hydrophobic DOX is more localized in the mitochondria than the nucleus and that more hydrophobic DOX is delivered into the mitochondria than into the nucleus than hydrophilic DOX·HCl.20, 37 When the nuclear or mitochondrial localization of the DOX delivered with the designed NPs was normalized by the CU, mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs, compared with free DOX, had an approximately 1.14-fold (i.e., 88% of that of free DOX) and 1.96-fold (i.e., 51% of that of free DOX) lower NLP, respectively, and an approximately 1.86-fold and 2.29-fold higher MLP than free DOX. In particular, when the respective MNP values for the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were calculated, they were 2.12-fold and 4.47-fold higher than that of free DOX, thus indicating that mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs


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delivered more DOX into the mitochondria than into the nucleus and that the dual functions of mitochondrial targeting and bioreducible degradation of the mPEG-(ss-TPP)2/DOX NPs allow for more efficient mitochondrial delivery of DOX than the single mitochondrial targeting function of mPEG-(TPP)2/DOX NPs, because more lipocationic TPP molecules are exposed on the surface of the NPs by the PEG shedding mechanism in the cytosol. Although the MNPs of the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs indicated their mitochondrial preference, their MNPs were not absolute values because the calculations were based on the relative values of free DOX. Thus, the flow cytometry results for the cellular and intracellular uptake of mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were supported by the confocal microscopy results of their intracellular location and intensity. The intracellular fluorescence intensity of the free DOX was much stronger than that of the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs (Figure 9(A) and Figure S1). Additionally, the mPEG-(ss-TPP)2/DOX NP-treated cells had a slightly higher DOX fluorescence intensity than that of the mPEG-(TPP)2/DOX NP-treated cells. The flow cytometric results (Figure 8(A)) and confocal microscopy results indicated that the CUs of the free DOX and drug-loaded NPs ranked in the following order: free DOX >> mPEG-(ss-TPP)2/DOX NPs > mPEG-(TPP)2/DOX NPs. Specifically, when considering the fluorescence of DOX (green) with the stained nucleus (blue) and mitochondria (red) together, we found that the majority of the free DOX was localized in the cytoplasm including the cytosol, the mitochondria, and other organelles, whereas a minor portion was detected in the nucleus. However, interestingly, the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs were much more localized in the cytoplasm than in the nucleus. Strong DOX fluorescence of mPEG-(ss-TPP)2/DOX NPs was detected in the mitochondria. Additionally, although the mPEG-(TPP)2/DOX NP-treated cells


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had quite a high DOX fluorescence in the mitochondria, their mitochondrial localization was lower than that of mPEG-(ss-TPP)2/DOX NPs. We then sought to determine whether the mPEG-(TPP)2/DOX NPs and mPEG-(ssTPP)2/DOX NPs actually existed in the nucleus, the mitochondria, or in both organelles. After the threshold level was crossed, the combined fluorescence of the delivered DOX, the stained nucleus, and the stained mitochondria was determined (Figure 9(B)). The results clearly indicated that the DOX molecules delivered by the mPEG-(TPP)2/DOX NPs, mPEG-(ssTPP)2/DOX NPs, and free DOX were more prevalent in the cytoplasm than in the nucleus and that more DOX molecules in the free DOX treatment reached the nucleus than the DOX molecules in the treatment with NPs. However, it was still difficult to determine whether the delivered DOX really existed in the mitochondria or the cytoplasm because the mitochondria are spread throughout the entire cytoplasm. Thus, because the nuclear and mitochondrial DOX had approximately a 10-fold lower fluorescence intensity than free DOX in other intracellular compartments, an additional analysis of the colocalization efficiency of DOX in the nucleus and the mitochondria was performed by using the analysis mode equipped in the confocal imaging software. As shown in Figure 9(C), the colocalization efficiency of free DOX in the nucleus was approximately 31%, whereas the mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs had approximately 18% and 9% in the nucleus, respectively. Interestingly, mPEG-(ss-TPP)2/DOX NPs had a much higher colocalization efficiency (85%) with the mitochondria than mPEG(TPP)2/DOX NPs (71%) and free DOX (58%). These results strongly support the trends of cellular and intracellular distribution of mPEG-(ss-TPP)2/DOX NPs as evaluated by flow cytometry.


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CONCLUSION In this study, we have developed novel mPEG-TPP conjugates with or without bioreducible disulfide bonds, namely mPEG-(TPP)2 and mPEG-(ss-TPP)2. On the basis of their lipophilic properties, the mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates self-assembled in aqueous solution to form NPs that are useful as bioactivatable nano-carrier systems to specifically target mitochondria. Our results showed that the bioreducible NPs have more effective DOX release profiles than their nonbioreducible counterparts under thiol-rich conditions (e.g., in the cytosol) because of cleavage of disulfide linkages in the mPEG-(ss-TPP)2/DOX NPs. In vitro subcellular distribution studies showed that the mPEG-(ss-TPP)2/DOX NPs have much more potential to target the mitochondria than free DOX and the mPEG-(TPP)2/DOX NPs. Further in vivo studies in a tumor xenograft model would provide details on the potential of this strategy to treat tumors.

Supporting Information Supporting Information contains size data by DLS and SEM measurements and sectional images of HepG2 obtained from confocal microscope at every 0.5 µm.

AUTHOR INFORMATION Co-corresponding authors *Prof. Kang Moo Huh Tel: +82-42-821-6663. Fax: +82-42-821-8910. E-mail: [email protected] *Prof. Han Chang Kang Tel: +82-2-2164-6533. Fax: +82-2-2164-4059.E-mail: [email protected]


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Author Contributions The manuscript was written with contributions from all of the authors. All authors have approved the final version of the manuscript. ‡ZK and YSC contributed equally to this study. Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2015R1D1A1A09056741) funded by the Ministry of Education, Science and Technology and the Industrial Technology Innovation Program [10060059, Externally Actuatable Nanorobot System for Precise Targeting and Controlled Releasing of Drugs] funded by the Ministry of Trade, Industry and Energy (MOTIE, Korea).

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Table 1. Synthesis results of mPEG-(TPP)2 and mPEG-(ss-TPP)2 conjugates.

Molecular weight Polymer

Feed molar ratio

Coupling ratio

(PEG:TPP)

(PEG:TPP)a

CAC

(g/mol)

(mg/mL)d Mn(NMR)

b

Mn(GPC)

c

mPEG-(TPP)2

1:6

1 : 1.52

2960.1

3659.8

0.0177

mPEG-(ss-TPP)2

1:6

1 : 1.41

2847.6

3723.8

0.0126

a

The coupling ratio of TPP to PEG was calculated from the peak integration of 1H-NMR spectra.

ba

The MW was calculated from the peak integration of 1H-NMR spectra.

c

The relative MW was determined via GPC measurements.

d

critical aggregation concentration (CAC) was determined via fluorescence intensity measurements

using pyrene as a fluorescent probe.


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Figure Captions Figure 1. Hypothetical scheme for DOX-loaded mPEG-(ss-TPP)2 NPs. During the self-assembly of mPEG-(ss-TPP)2 conjugates, hydrophobic and anticancer DOX drugs are encapsulated in the hydrophobic core of mPEG-(ss-TPP)2 NPs. In the thiol-rich cytosol, the bioreducible mPEG-(ssTPP)2/DOX NPs are transformed into TPP/DOX NPs via disulfide dissociation-induced mPEG detachment from the NPs. TPP/DOX NPs specifically target the mitochondria, thereby providing effective therapeutic effects of the delivered DOX.

Figure 2. Synthesis and characterization of the mPEG-(TPP)2 conjugate and the mPEG-(ssTPP)2 conjugate: (A) Synthetic scheme, (B) 1H-NMR spectra, and (C) FT-IR spectra of the designed conjugates.

Figure 3. Size characteristics of mPEG-(TPP)2/DOX NPs and mPEG-(ss-TPP)2/DOX NPs in BSA-free PBS, BSA (10%)-containing PBS, or a dry condition: (A, B) dynamic light scattering (DLS)-based size distributions of the NPs in BSA-free PBS, (C, D) scanning electron microscope (SEM)-based sizes of the dried NPs, and (E) time-dependent size changes of the NPs in BSA (10%)-containing PBS.

Figure 4. Time-dependent changes in the size distribution of mPEG-(TPP)2/DOX NPs (A and B) and mPEG-(ss-TPP)2/DOX NPs (C and D) in PBS without (A and C) and with (B and D) DTT (10 mM), respectively.


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Figure 5. Thiol-dependent DOX release profiles from mPEG-(TPP)2/DOX NPs and mPEG-(ssTPP)2/DOX NPs in either DTT-free PBS or DTT (10 mM)-containing PBS at 37 °C. The data are expressed as the mean ± SE (n = 5). (*mean p < 0.05).

Figure 6. In vitro cytotoxicity of mPEG-(TPP)2 NPs and mPEG-(ss-TPP)2 NPs in (A) HepG2 and (B) HeLa cells after 48 h of co-incubation. The data are expressed as the mean ± SE (n = 5).

Figure 7. In vitro DOX effects of mPEG-(TPP)2/DOX NPs, mPEG-(ss-TPP)2/DOX NPs, and free DOX in HepG2 (A) and HeLa (B) cells 48 h after treatment. The data are expressed as the mean ± SE (n = 5).

Figure 8. (A) The cellular uptake (CU) nuclear uptake (NU), and mitochondrial uptake (MU) and (B) the nuclear localization preference (NLP), mitochondrial localization preference (MLP), and mitochondria-to-nucleus preference (MNP) of DOX delivered by mPEG-(TPP)2/DOX NPs, mPEG-(ss-TPP)2/DOX NPs, and free DOX in HepG2 cells 4 h after treatment ([DOX] = 3 µg/mL). A representative flow cytometric histogram was used, and the quantitative data are expressed as the mean ± SE (n = 3). (* and ** mean p < 0.05 and P < 0.5, respectively).

Figure 9. (A) The intracellular distribution (from center-sectioned confocal images), (B) the fluorescence intensity profile (of a representative line [the white arrow in (A)]), and (C) the colocalization efficiency of DOX delivered by mPEG-(TPP)2/DOX NPs, mPEG-(ss-TPP)2/DOX NPs, and free DOX in HepG2 cells 4 h after treatment ([DOX] = 1 µg/mL). (*mean p < 0.05).


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Figure 1.


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Figure 2.


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Figure 3.


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Figure 4.


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Figure 5.


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B 120

100

100

80 60 mPEG-(TPP) 2 NP mPEG-(ss-TPP) 2 NP

40 20

Cell viability (%)

A 120 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

Biomacromolecules

HepG2

60 40 20

HeLa

0 1

80

10 100 Concentration Material (µ µ g/mL)

1000

mPEG-(TPP)2 NP mPEG-(ss-TPP)2 NP

0

1 10 100 Concentration Material (µ µg/mL)

1000

Figure 6.


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A 100

HepG2

80 60 40 20 0 0.01

B 100

Cell viability (%)

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

Free DOX mPEG-(TPP)2/DOX NP mPEG-(ss-TPP)2/DOX NP

Page 42 of 45

HeLa

80 60 40 20 0

0.1 1 [DOX] (µ µg/mL)

10

0.01

0.1 1 [DOX] (µ µg/mL)

10

Figure 7.


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Figure 8.


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C

Colocalization efficiency (%)

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

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100

80

Nuclei Mitochondria Others

60

40

20

0 Free DOX

mPEG-(TPP)2

mPEG-(ss-TPP)2

/DOX NP

/DOX NP

Figure 9


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Table of Contents (TOC) graphic


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Bioreducible Poly(ethylene glycol ... - ACS Publications

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