Intracellular NO-Releasing Hyaluronic Acid-Based Nanocarriers: A

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Biological and Medical Applications of Materials and Interfaces

Intracellular NO-Releasing Hyaluronic Acid-Based Nanocarriers: A Potential Chemosensitizing Agent for Cancer Chemotherapy DaEun Kim, Chan Woo Kim, HONG JAE LEE, Kyung Hyun Min, Kyu Hwan Kwack, Hyeon-Woo Lee, Jaebeum Bang, Kiyuk Chang, and Sang Cheon Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06848 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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ACS Applied Materials & Interfaces

Intracellular NO-Releasing Hyaluronic AcidBased Nanocarriers: A Potential Chemosensitizing Agent for Cancer Chemotherapy

Da Eun Kima‡, Chan Woo Kimb‡, Hong Jae Leea, Kyung Hyun Mina, Kyu Hwan Kwackc, Hyeon-Woo Leec, Jaebeum Bangd, Kiyuk Chang b,*, Sang Cheon Leea,*

a

Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea

b

Cardiovascular Center and Cardiology Division, Seoul St Mary's Hospital, The Catholic University of Korea, Seoul 06591, Republic of Korea

c

Department of Pharmacology, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea

d

Department of Dental Education, School of Dentistry, Kyung Hee University, Seoul 02447, Republic of Korea

ACS Paragon Plus Environment


ABSTRACT 2500

PBS Free DOX GSNO-HANPs GSNO-HANPs + DOX

CD44

Receptor binding

Hyal-1

Ascorbic acid (a reducing agent)

. . ] . ] ]

. ].

. ]

. .] ]

] Facilitated NO release

Tumor volume (mm3)

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2000 1500 1000 500 0

Intracellular degradation by Hyal-1

0

3

6

9

12

15

Days

In this work, we investigate whether S-nitrosoglutathione (GSNO)-conjugated hyaluronic acid-based self-assembled nanoparticles (GSNO-HANPs) can be useful as a chemosensitizing agent to improve the anticancer activity of doxorubicin (DOX). The GSNO-HANPs were prepared by aqueous assembly of GSNOconjugated HA with grafted PLGA (poly(lactide-co-glycolide)). Aqueous GSNO stability shielded within the assembled environments of the GSNO-HANPs was greatly enhanced, compared to that of free GSNO. NO release from the GSNOHANPs was facilitated in the presence of hyaluronidase-1 (Hyal-1) and ascorbic acid (AsA) at intracellular concentrations. Microscopic analysis showed GSNOHANPs effectively generated NO within the cells. We observed NO made the human MCF-7 breast cancer cells vulnerable to DOX. This chemosensitizing activity was supported by the observation of an increased level of ONOO(peroxynitrite), a highly reactive oxygen species, upon co-treatment with the GSNO-HANPs and DOX. Apoptosis assays showed that GSNO-HANP alone exhibited negligible cytotoxic effects and reinforced apoptotic activity of DOX. Animal experiments demonstrated the effective accumulation of GSNO-HANPs in solid MCF-7 tumors and effectively suppressed tumor growth in combination with DOX. This hyaluronic acid-based intracellularly NO-releasing nanoparticles may serve as a significant chemosensitizing agent in treatments of various cancers. KEYWORDS: nitric oxide, hyaluronic acid, intracellular delivery, doxorubicin, chemosensitizing effect,

1


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1. INTRODUCTION

Nitric oxide (NO) is a key chemical messenger exerting numerous bioregulatory actions in cellular signalling, vascular modulation, neurotransmission, and cell proliferation or apoptosis.1-4 In particular, NO has been recognized as a significant controlling factor in suppressing tumor growth.5,6 It is well documented that NO alone expresses a pro-apoptotic activity in cancer therapeutics, but only at high doses.7 Owing to the difficulty of achieving high doses in vivo conditions, there exists a limitation in the use of NO alone. It was previously demonstrated that cotreatment of NO with various antineoplatic agents, including cisplatin and DOX, resulted in enhanced anticancer effect.8-10 A prerequisite enabling this chemosensitizing effect of NO for cancer treatments is a delivery of NO within the cytosol of cancer cells at a high efficacy. However, the intracellular delivery of NO at the molecular level is a problematic issue, because NO is strongly reactive, and plasma half-life is less than a few seconds.11 Recently, several classes of NO donors, including S-nitrosoglutathione (GSNO), nitrosoamines, metal-NO complexes, organic nitrates, and N-diazeniumdiolates, have been developed to stably store and release NO.12-18 However, NO donors are not extensively utilized due to their low stability (uncontrolled NO release), inefficient intracellular NO transport, and toxicity issues. To improve the stability of these NO-donating species, various NO 2


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donors have been conjugated or loaded to organic or inorganic particles, including polymeric

assemblies,

silica

nanoparticles,

and

surface-modified

Au

nanoparticles.19-24 Of the diverse set of NO donors, GSNO has an advantage for intracellular NO delivery in that it is an endogenous species and does not generate toxic byproducts after liberating NO species, a problem that is frequently reported with N-diazeniumdiolates and NO-metal complexes.12,19 GSNO also suffers from insufficient structural stability in water. In addition, the delivery efficacy of GSNO to the cells of target tumors is very low.20-22 For this reason, it is a challenging subject to design an intracellular vehicle that efficiently overcome these inherent limitations of GSNO. Recently, our group reported in vitro proof-of-concept results preliminarily demonstrating that GSNO-incorporated inorganic biomineralinspired nanocarriers induced facilitated generation of NO within cells, which sensitized human breast cancer cells to DOX therapy.24 Herein, we develop a novel type of GSNO-conjugated hyaluronic acidbased organic self-assembled nanoparticle (GSNO-HANP) that can stably store GSNO species. We examine that the GSNO-HANPs effectively protect GSNO species from aqueous degradation and trigger preferentially NO production by hyaluronidase (Hyal-1) in cytosols and intracellular reducing environments. Our

3


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primary goal is to identify the GSNO-HANPs as a useful in vivo chemosensitizing agent for improving chemotherapeutic effect of DOX. As shown in Figure 1, we illustrate the overall process for fabrication of the GSNO-HANPs, intracellular NO generation, and the chemosensitizing effects of the released NO for DOX treatments. The aqueous assembly of GSNO-cotaining PLGA-grafted HAs led to the formation of the GSNO-HANPs (Figure 1a). Upon entering the cancer cells, the GSNO-HANPs are degraded by Hyal-1 of an elevated level within the cells, thereby liberating GSNO in the cytosols and permitting its reduction by intracellular ascorbic acid (AsA) (Figure 1b). In this work, the aqueous stability of the conjugated GSNO in the GSNOHANPs was compared to that of free GSNO. The in vitro monitoring of NO generation was performed at cellular interior and exterior pH, and AsA concentrations with or without Hyal-1. Receptor-mediated cellular uptake of the GSNO-HANPs and the intracellular NO liberation were visualized using a confocal microscope. Using the MCF-7 cells, we examined the chemosensitizing activity of the pretreated GSNO-HANPs for DOX therapy. To support the chemosensitizing ability of the GSNO-HANPs for DOX therapy, the intracellular formation of ONOO- was monitored. An apoptosis assay for the GSNO-HANPs-induced chemosensitizing effect was also performed. In vivo antitumor activity of DOX pretreated with the GSNO-HANPs was also evaluated using solid MCF-7 tumors. 4



(a)

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(b) CD44

Receptor binding GSNO-PLGA-HA copolymer

Self-assembly

Hyal-1

Ascorbic acid (a reducing agent)

. . ] . ] ]

GSNO-HANP

. ].

]

. ]

. .] ]

Intracellular degradation by Hyal-1

Facilitated NO release

(c)

NO release O2

O2·NO

DOX·-

DOX

O=NOO(peroxynitrite)

Mitochondria

Cyt C Nucleus

Apoptosis

Figure 1. a) Self-assembly of the GSNO-PLGA-HA copolymer for GSNO-HANPs, b) NO release from GSNO-HANPs triggered by intracellular Hyal-1 and ascorbic acid, and (c) postulated mechanism of chemosensitizing effect of GSNO-HAPNs for DOX therapy (production of ONOO- as a key element for NO-induced chemosensitizing effect).

5


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2. EXPERIMENTAL SECTION 2.1. Materials. PLGA (MW: ~ 10,000 Da, 80:20 of lactide:glycolide) was supplied by Polysciences, Inc. (Warrington, PA). HA (MW: 10,000~20,000 Da) was supplied by Lifecore Biomedical (Chaska, MN). DOX hydrochloride was supplied by from BoRyung Inc. (Seoul, Korea). GSNO, fluorescein isothiocyanate isomer I (FITC), sodium nitrite, ascorbic acid (AsA), hylauronidase-1 (Hyal-1), 4dimethylaminopyridine

(DMAP),

poly(ethylene

glycol)

dimethyl

ether

(CH3O(CH2CH2O)nCH3 (MW: 2,000 Da)), and N,N’-dicyclohexylcarbodiimide (DCC) were supplied by Sigma Co. (Milwaukee, WI). 2.2. Synthesis and characterization of the GSNO-conjugated PLGA-grafted hyaluronic acid copolymer (GSNO-PLGA-HA). The GSNO-PLGA-HA copolymer was synthesized by a modification of a literature proceure.25 In brief, the complex powder of HA/poly(ethylene glycol) dimethyl ether was dissolved in DMSO of 10 mL at 70 oC. Poly(ethylene glycol) dimethyl ether was the nanocomplex agent for HA solubilization in DMSO.25 After addition of PLGA (262 mg) in DMSO (5 mL), DCC and the catalytic amout of DMAP were added. After 6 h of stirring, addition of GSNO (528 mg) was followed. After 12 h, the solution was then dialyzed using an excess amount of DMSO for 1 day with a dialysis membrane (Molecular weight cut-off: 10,000 g/mol) to remove reaction by-products including

6



dicyclohexylurea. To further remove any unreacted PLGA and polymeric byproducts, such as possibly formed self-conjunctive PLGA, the dialyzed mixture was first precipitated into acetone, a good solvent for PLGA. Finally, GSNOPLGA-HA was collected by the precipitation from DMSO into cold diethyl ether (× 3). The isolated GSNO-PLGA-HAs (yield = 70%) were dried at room temperature in vacuo and stored at 4 oC in the dark. The conjugation of GSNO and PLGA to the backbone of HA was confirmed by 1H NMR at 400 MHz (Varian INOVA400, USA). The percentages of the grafted PLGA and the conjugated GSNO to the repeating unit of HA were calculated by 1H NMR analysis. The conjugated GSNO content in the GSNO-PLGA-HAs was also determined by quantification of NO in GSNO moieties. To completely dissociate NO molecules, the aqueous solution of the GSNO-PLGA-HAs were treated with Hyal-1 (120 units) and AsA (5 mM) until the NO concentration dose not further increase. The dissociated NO were quantified by the Griess assay kit (G-7921) (Molecular Probes, USA) by comparing with the standard sodium nitrite solution (1~100 μM). The UV-vis spectra of the GSNO-PLGA-HA copolymer was recorded on a UV-1650PC spectrometer (Shimadzu, Japan). The ATR-FTIR spectroscopic analysis was performed using a FTIR equipment (Tensor 27, Bruker, Germany). 2.3. Fabrication and characteristics of the GSNO-HANPs. The GSNO-

7


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PLGA-HAs in DMSO (10 mg/mL) was placed within a membrane bag, and then the dialysis proceeded using an excees water for 24 h (Molecular weight cut-off of the dialysis membrane: 25,000 g/mol). The GSNO-HANPs (yield = 93%) were collected by freeze-drying, and stored at 4 oC under protecting the product from light. We estimated CMC (critical micelle concentration) for GSNO-PLGA-HA by pyrene excitation spectra obtained from a fluorometer (JASCO FP-6500).26 The concentrations of GSNO-PLGA-HA copolymer are from 5 × 10-5 to 0.1 mg/mL ([pyrene] = 6.0 × 10-7 M). The wavelengh ranged 280~360 nm, and em was fixed at 395 nm. We detrermined CMC at a concentration, where dramatic shift in pyrene (0,0) bands was observed.26 Dynamic light scattering (DLS) analysis was utilized to obtain an information for mean size in water, distribution in particle sizes, and polydispersity factors (μ2/Γ2) of the GSNO-HANPs (90 Plus, Brookhaven Co., New York, NY).27 The shape of the GSNO-HANPs was estimated using a transmission electron microscope (JEM-2000EX, JEOL). The nanoparticle samples deposited on a TEM grid were negatively stained with an aquesous solution of uranyl acetate (1 %) before analysis.28

2.4. Stability of GSNO conjugated in the HANPs. Stability of GSNO conjugated in HANPs was estimated by evaluating the degree of NO dissociation from the GSNO moieties using the Griess assay. In detail, the GSNO-HANPs (3 8



mg/mL) within a dialysis bag was placed in the PBS solution (10 mL), and maintained at 37 °C. Sample solutions (150 μL) in the dialysis bag were collected at fixed time periods and were combined with an aqueous solution containing Griess reagent of 30 μL. After incubation for 30 min, the samples was assessed using a wavelength of 548 nm (Benchmark™ Plus Microplate Reader). The sodium nitrite solution of 1~100 μM was used as a standard sample. Stability of GSNO conjugated in HANPs was compared to that of the native GSNO molecules and GSNO conjugated in HA (GSNO-HA), which was synthesized through a similar procedure used for the synthesis of the GSNO-PLGA-HA copolymer except PLGA addition. 2.5. Controlled NO release from the GSNO-HANPs. In vitro NO release from the GSNO-HANPs under in extracellular and intracellular conditions considered in terms of pH, the concentration of AsA, and the presence or absence of Hyal-1. For extracellular conditions, the medium of the PBS solution (pH 7.4) containing rather lower concentration of AsA (80 μM) was used, and for intracellular condition, the medium of the PBS solution (pH 5.0) containing AsA (5 mM) and Hyal-1 (120 units). A membrane bag containing the aqueous solution of the GSNO-HANPs (5 mg/mL) was placed in each release medium (10 mL) for the release experiment. At fixed time periods, the release medium was mixed with an aqueous solution containing Griess reagent (30 L). The nitrite absorption was 9


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obtained using a waveleng of 548 nm. Nitrite concentrations were finally converted to the concentration of the cumulative NO release. 2.6. Cellular uptake of the GSNO-HANPs. Korean Cell Line Bank provided the MCF-7 cells. For experiments for cellular uptake, a green-fluorescent FITClabeled GSNO-HANPs were used. The GSNO-HANPs were chemically modified using adipic dihydrazide (ADH) to provide primary amine groups for FITC labeling, which was based on the literature process of HA modification.29 The simple mixing of amine-modified GSNO-HANPs with FITC resulted in FITClabeled GSNO-HANPs (yield = 90%). The MCF-7 cells were seeded (1 × 105 cells/well) in a dish (SPL Life Sciences) in RPMI 1650 (Gibco BRL) containing penicillin-streptomycin (1%, Gibco-BRL) and heat-inactivated fetal bovine serum (10%) and incubated for 24 h under humidified conditions. FITC-labeled GSNOHANP solutions (200 μg/mL, 1 mL) and LysoTracker (50 nM) were than treated. After incubation, the fixation was done using a formaldehyde (3.7 %). The distribution of the GSNO-HANPs in cytosols was visualized using a confocal microscope (CLSM, C1si, Nikon). To verify the CD44 receptor-mediated uptake of GSNO-HANPs, fluorescent Cy5.5 was labeled to GSNO-HANPs. As described in the procedure of FITC labeling to the GSNO-HANPs, ADH was used to produce the primary amines in the HA backbone for the reaction with a hydroxysuccinimide ester of Cy5.5 NHS.29 For the CD44 receptor competitive inhibition study, the MCF-7 cells were pretreated for 60 min with the medium containing free HA (1 10



mL, 5 mg/mL). To assess the effect of free HA existence, the cells were incubated with Cy5.5-GSNO-HANPs (200 μg/mL) for 1 h. After cell fixation with formaldehyde solution (4%), CLSM was the performed for visualization of cellular uptake. 2.7. Visualization of intracellular NO generation. NO intracellularly relea sed from the GSNO-HANPs was visualized by detecting fluorescent adduct s of NO/DAF-FM (Molecular Probes Int.) and NO. The cells of 5 × 104 cells/well in glass dishes were incubated for 24 h and further incubated with the solution of DAF-FM diacetate (2 mL) for 40 min. The GSNO-HANPs dispersed in medium (2 mL, 500 μg/mL) were added to the culture dish. After fixation, Alexa Fluor 594-labeled wheat germ agglutinin (2 mL, 5 μg/mL) was used for staining of the cell membrane, and, for nuclei staining, we used DAPI (4′,6-diamidino-2phenylindole). The culture dish was washed (× 3) with the PBS, and florescence images of MCF-7 cells were obtained using a CLSM (C1si, Nikon): NO/DAF-FM adduct (Ex./Em. wavelengths= 495/515 nm), Alexa Fluor 594-conjugated WGM (591/618 nm), and DAPI (350/461 nm), respectively.24 2.8. Cytotoxicity of the HANPs and the GSNO-HANPs. The cells of 5 × 103 cells/wells were incubated with the HANPs or GSNO-HANPs at 37 °C (5% CO2). GSNO-HANPs with various concentrations were used (10 ~ 1,000 μg/mL). And then, we added a cell counting kit-8 solution to the culture media. The viable 11


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cells were assessed by measuring absorbance at a wavelength of 450 nm. 2.9. Chemosensitizing effect by GSNO-HANPs. MCF-7 cells were pretreated with each of the GSNO-HANPs (600 μg/mL ) and the HANPs (600 μg/mL) for 24 h at 37°C. And then, DOX (0.01~1 μg/mL) of 100 μL was added. After 48 h, a a cell counting kit-8 solution was used for estmation of viable cells. 2.10. Apoptosis assay for chemosensitizing activity of GSNO-HANPs. To evaluate apoptotic activity, the MCF-7 cells were incubated with GSNO-HANPsdispersed media (300 and 600 μg/mL). After pre-treatment of the GSNO-HANPs, 100 μL medium containing 0.12 μg/mL of DOX was added. After collecting cells by trypsinization and subsequent centrifugation, the suspension of the cells was prepared using 1X Binding Buffer. The cell suspension was then treated with PI (propidium iodide) and FITC-labeled Annexin V of an equal volume of 5 μL. After 15 min, the cells were assessed using flow cytometry analysis (BD FACSCaliburTM, CA) to quantify apoptosis. Flow cytometry were detected at emission/excitation wavelengths of 530/488. The image of apoptotic cells was obtained by CLSM. 2.11. Quantification of intracellular peroxynitrite formation. To detect the ONOO- at intracellular environments, Cell MeterTM Assay Kit (AAT Bioquest, USA) for peroxynitrite assay was used. The 5 × 103 cells/well were treated with medium containing GSNO-HANPs (100 μL) for 24 h. DOX-dissolved medium (100 μL,

12



concentration of 0.12 μg/mL, IC50 of DOX co-treated with GSNO-HANPs) was then added and incubated for 12 h. After addition of DAX-J2TM PON Green working solution (10 %) and incubation for 1 h. The absorbance of each well was monitored at Ex./Em. = 490/530 nm. 2.12. In vivo and ex vivo imaging and biodistribution analysis. All procedures of in vivo animal experiment were performed in a permission by a Committee of Institutional Anima Care and Use. In vivo tumor models were prepared by subcutaneously injecting 1 × 107 MCF-7 cells in a mixture of PBS/Matrigel with an equal volume at the right flank region of athymic nude mice (female 6-week-old). For volume calculation of solid tumors, we employed an equation of (tumor length) × (tumor width)2/2. When tumor volume grew to 100150 mm, free Cy5.5 (0.1 mg/kg in 100 µL of PBS) and Cy5.5-labeled GSNOHANPs (2 mg/kg, 0.1 mg/kg of Cy5.5 in 100 µL of PBS) were injected intravenously, respectively (n = 3 per each group). After injection of samples, the mice were imaged under an NIRF imaging equipment (IVIS Lumina XRMS, PerkinElmer, USA) with the standard filter set of Cy5.5 at different time points. After sacrifice of mice, we collected tumors and major organs. All imaging analyses were performed with an IVIS imaging software (Living Imaging, PerkinElmer, USA). For ex vivo fluorescence imaging, the dissected tumors were treated in the tissue freezing medium (- 80 °C). Sliced tumor tissues (5 µm) were 13


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stained with DAPI and observed with CLSM (ZEISS LSM 510, Carl Zeiss) under HeNe 5 mW 633 nm laser. 2.13. In vivo antitumor efficacy. When tumors grown in athymic nude mice reached to 80‒100 mm3, The mice were prepared for four different sample groups (n = 5), and were injected through tail veins with the following samples (100 µL): (i) the PBS solution, (ii) free DOX, (iii) GSNO-HANPs, and (iv) GSNOHANPs with an interval of 1 h. The injected dose of DOX and GSNO-HANPs in three sample groups are 2.5 and 5.0 mg/kg, respectively. The treatments were given once every 3 days for 9 days. The mice were euthanatized at over 2,000 mm3 of tumor volume, which was considered as the experimental endpoint according to the animal protocols. For histological analysis, tumor tissues and major organs were fixed using paraformaldehyde (4%). All tumor tissues or organ-containing blocks embedded in paraffin were sectioned at a thickness 5 µm. The staining of major organs was carried out using hematoxylin and eosin (H&E). A slide scanner (SCN400, Leica Microsystems, Germany) was utilized for analysis of stained organs. The tumor tissues were evaluated by TUNEL immunofluorescent analysis with TdT in situ apoptosis detection kit (TREVIGEN, USA) and DAPI staining using CLSM. The apoptosis rate of TUNEL assay was calculated by image analysis software (ImageJ,

14



NIH) and calculated with an equation of (# of apoptotic cells / total # of cells) × 100%. 2.14. Statistics. Based on Student’s t-test, we estimated a significance of statistics in data from control and experimental samples. We considered P values < 0.05 as the significance of statistics.

3. RESULTS AND DISCUSSION

3.1. Characteristic analyses for GSNO-PLGA-HA. Hyaluronic acid containing grafted PLGA and conjugated GSNO was synthesized through carbodiimide-mediated coupling chemistry (Figure S1 in Supporting Information). Conjugation of GSNO to the PLGA-grafted HA copolymer (PLGA-HA) was verified by 1H NMR spectroscopy (Figure S2 in Supporting Information). GSNO conjugation into PLGA-HA was found successful by observing a resonance peak at 8.24 ppm (signal f), which was ascribed to the proton of the newly formed amide bond (-(C=O)NH-) newly formed between GSNO and HA (Figure S2b in Supporting Information). The percentages of PLGA grafting and GSNO conjugation to the repeating unit of HA, were 3.1 % and 3.0 %, respectively (Table 1). In addition, the conjugated percentage of GSNO was also confirmed by quantification of completely liberated NO molecules from GSNO in the presence

15


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of Hyal-1 (120 units) and AsA (5 mM). Using ATR-FTIR, we found the existence of GSNO in PLGA-HA structures. The GSNO-PLGA-HA copolymer exhibited SNO absorption signals, which originates from GSNO at 1250 and 1460 cm-1 (Figure S3 in Supporting Information).23 UV-Vis spectroscopy supported the GSNO conjugation to the PLGA-HA structure, showing the characteristic absorption of SNO bonds at 340 nm (Figure S4 in Supporting Information).30

Table 1. The percentages of grafted PLGA and conjugated GSNO to the repeating unit of HA.

a

copolymer

percentage of grafted PLGAa (%)

percentage of conjugated GSNOa,b (%)

GSNO-PLGA-HA

3.1

3.0

Calculated using 1H NMR spectra. bCalculated using 1H NMR spectra and the Griess assay.

3.2. Fabrication and characteristics of GSNO-HANPs. The GSNO-PLGA-HA copolymer contain anionic HA and hydrophobic PLGA as a main component, and thus, the copolymer can undergo assembly in water to generate a nanoparticle structure with PLGA-rich inner domains and hydrated HA outer domains. We hypothesize that the conjugated GSNO moieties may have a higher probability of residing around the PLGA-rich assembled inner core domains, because the inner domains of the assembled nanoparticles occupy a much larger volume than the outer surfaces of the hydrated outer domains. To support our assumption, we need 16



to provide more information about where the majority of GSNO is located in the nanostructure of GSNO-HANPs. This issue will be discussed in the following section describing the stability of GSNO conjugated in assembled HANPs. The self-assembly of the GSNO-PLGA-HA copolymer was verified by determining the critical micelle concentration (CMC) in aqueous phase. For CMC measurement, we employed a photophysical property of pyrene excitation spectra. It is well documented that, upon assembly of polymeric amphiphiles, polyaromatic hydrophobic pyrene tends to reside preferably within the hydrophobic core domains, which resulted in a (0,0) peak shift to the range of longer wavelength in pyrene excitation spectra.26,31 We observed this characteristic change in pyrene spectra from 337 nm to 339 nm as the aqueous concentration of the copolymer increased (Figure S5a in Supporting Information). The CMC was calculated by the intersection between two lines drawn from the experimental data at the lower and the higher concentrations (Figure S5b in Supporting Information). The CMC value of the GSNO-PLGA-HA copolymer was estimated to be 4.5 g/mL. This low CMC is similar to those values observed for previously reported amphiphilic graft copolymers.32,33

Using TEM analysis, we found the GSNO-HANPs have a spherical shape (Figure S6a in Supporting Information). The mean diameter of the GSNO-HANPs 17


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calculated by DLS was 264.5 ± 7.9 nm with a moderate size distribution (Figure S6b in Supporting Information and Table 2). The size of GSNO-HANPs estimated by DLS was rather larger compared to the one visualized by TEM. For TEM measurement, the samples should be dehydrated on a copper grid. A number of literature works for TEM analysis reported that the collapse of the hydrated shell or surface of organic and inorganic nanoparticles under the high vacuum condition.34,35 This analytical condition for TEM samples often leads to structural distortions and reduction in size, compared to the hydrated state, which was estimated by DLS.

Zeta potential (of GSNO-HANPs was estimated -16.3 ± 1.0 mV, due to the anionic hydrated layers of the HA outer shells.

Table 2. Characteristics of GSNO-HANPs in the aqueous phase.

nanoparticle

mean diameter (nm)

 (mV)

μ2/Γ

GSNO-HANPs

264.5 ± 7.9

-16.3 ± 1.0

0.27 ± 0.05

3.3. Stability of GSNO conjugated in HANPs and controlled NO release. A major weakness of S-nitrosothiol class compounds including GSNO is known as their poor aqueous stability.23,36 We are interested in knowing whether the 18



assembled structure of the GSNO-HANPs can improve water stability of GSNO. Figure 2a exhibited the NO liberation behavior from native GSNO and GSNOHANPs. Native GSNO decomposed to a level of 30.5 % at 72 h by continuously releasing NO, which demonstrates the poor aqueous stability of GSNO. In contrast, the conjugated GSNO in the assembled GSNO-HANP nanoparticles exhibited a significantly improved stability. At 72 h, 83.3 % of the GSNO was maintained. In addition, there was almost no dissociation of GSNO after 12 h. The initial 17.7 % of decomposition may be ascribed to GSNO located on the GSNO-HANP surface. Interestingly, GSNO-HA, a water-soluble HA containing simply conjugated GSNO, exhibits a negligible improvement in stability of GSNO, compared with native GSNO. As expected, GSNO-HA cannot undergo self-assembly in water, and thus almost all conjugated GSNO moieties are exposed to the aqueous phase. This may lead to the low stability of GSNO conjugated in HA. This strongly indicates that the main contributing factor to increase the stability of GSNO is the formation of the core domain by self-assembly of GSNO-HANPs. Hence, it can be suggested that the majority of GSNO moieties are located within the PLGA-rich inner domains of the assembled GSNO-HANPs. The rigidity of densly packed PLGArich inner domains of the assembled GSNO-HANPs may result in a significant improvement in GSNO stability.

19


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In our system, the intracellular NO release from the GSNO-HANPs may be facilitated by the following two steps: (i) rapid degradation of the HA backbones of the GSNO-HANPs by Hyal-1, which exists at an elevated level within the tumor cells37,38, to enable the exposure of GSNO in the cytosol, and (ii) the liberation of NO from exposed GSNO through its reduction by AsA. To prepare in vitro environments, the endosomal pH (5.0)/the intracellular reducing condition (5 mM of AsA) were employed, whereas for the extracellular condition, we chose a physiological pH (7.4) and 80 M AsA.39,40 As shown in Figure 2b, under extracellular conditions, the GSNO-HANPs effectively inhibited NO release. After 24 h, the percentage of NO release was as low as 12.0 %. In contrast, under intracellular conditions (pH 5.0 and AsA of 5 mM), NO was released rapidly, and the % NO release was considerably higher, reaching 75.4 %. This result indicates that intracellular AsA, at high concentrations, can penetrate into the assembled structures and effectively reduce GSNO by cleaving S-NO bonds. Notably, the additional presence of Hyal-1 in the intracellular conditions resulted in faster NO release than occurred in the release media lacking Hyal-1. In the presence of additional Hyal-1, the NO release from the GSNO-HANPs reached 95.8 %. Taken together, the results of the in vitro release assay supported our postulated mechanism of intracellular NO generation within tumor cells, which was suggested in Figure 1b.

20



(a) GSNO Amount (%)

100

80

60

40

GSNO GSNO-HANPs GSNO-HA

20

0 0

20

40

60

Time (h)

(b) 100

NO Released (%)

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

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80

60

40 pH 7.4 + 80 M AsA pH 5.0 + 5 mM AsA pH 5.0 + 5 mM AsA + Hyal-1

20

0 0

5

10

15

20

25

Time (h) Figure 2. (a) Stability of GSNO, GSNO-HANPs, and GSNO-HA in PBS and (b) in vitro behavior of NO generation from the GSNO-HANPs (n=3).

3.4. Uptake behavior of GSNO-HANPs in MCF-7 cells. For cellular uptake 21


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experiments, the red-fluorescent LysoTracker was used to label acidic compartment within the MCF-7 cells. The GSNO-HANP-treated cells exhibited strong green fluorescence in acidic compartments (1 h incubation) (Figure S7 in Supporting Information). After incubation for a further 5 h, the green fluorescence became stronger. This indicates efficient uptake of FITC-labeled GSNO-HANPs by MCF-7 cells. We carried out an experiment to support that cellular internalization of GSNO-HANPs occurred via a receptor-mediated pathway. It is noteworthy the pretreatment of the cells with free HA significantly lowers the level in cellular uptake of GSNO-HANPs (Figure S8 in Supporting Information). This indicates GSNO-HANP transport within MCF-7 cells was mediated by the binding of GSNO-HANPs to the HA receptor, CD44. When GSNO-HANPs are used as intravenous carriers for tumor treatment, a high delivery efficacy of the GSNOHANPs may be obatined by CD44-mediated endocytosis.29,37,41 3.5. Visualization of intracellular NO release. Next, we examined whether the GSNO-HANPs can effectively generate NO molecules within the cytosol. To verify NO release in intracellular environments, DAF-FM DA was employed for NO assay. It is rapidly degraded by esterases upon cellular uptake to form DAFFM, which generates a green fluorescent benzotriazole derivative when reacted with NO (Figure S9a in Supporting Information).42 After pretreatment with DAF-

22



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FM DA, GSNO-HANPs (300 μg/mL) was added to the MCF-7 cells. After 6 h, NO production in cytosols were observed by CLSM.

DAPI

DAF-FM DA

Alexa 594

superimpose

Negative control

DAF-FM DA (+)

DAF-FM DA (+) GSNO-HANPs (+)

Scale bar : 20 m

Figure 3. CLSM images of intracellular NO release from GSNO-HANPs. Figure 3 exhibits that the cell group (DAF-FM DA (+)) displayed a low level of green fluorescence due caused by native NO in the cytosol. In contrast, the GSNOHANPs-treated cells emitted bright green fluorescence. These results confirm efficient intracellular NO generation by the GSNO-HANPs. In addition, the amount of NO complexed with DAF-FM tended to gradually increase with time (Figure S9b and c in Supporting Information).

23


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3.6. In vitro chemosensitizing effect of GSNO-HANPs. Next, we were interested in knowing whether NO-liberating GSNO-HANPs can promote the anticancer effect of DOX by inducing a chemosensitizing effect. Previously, various possible mechanisms have been suggested to explain this chemosensitizing effect.8,43,44 In the initial step of one plausible mechanism, the DOX semiquinone radical is generated, which reacts with O2 and finally leads to the elevation of O2•(superoxide) levels.45 It is expected that NO liberated by the GSNO-HANPs reacts with O2•- to increase the level of ONOO- (peroxinitrite), a highly reactive oxygen species.46 ONOO- can induce a significant damage to main intracellular sites, such as mitochondria and nucleus, which are probably be more vulnerable to DOX. Based on this NO-related intracellular event, we postulated a mechanism through which GSNO-HANP-sensitization improves DOX efficacy, as illustrated in Figure1c. We found that the cytotoxicity of GSNO-free HANPs and GSNOHANPs is negligible up to 1,000 g/mL (Figure S10 in Supporting information). This reflects the GSNO-HANPs did not release a sufficient level of NO to induce cell toxicity. As shown in Figure 4, co-treatment with GSNO-free HANPs and DOX (HANPs + DOX) inhibited cell proliferation to a level similar to that of DOX alone. It is of interest to note that pretreatment of the cells with GSNO-HANPs (600 g/mL) notably promoted the anticancer effect of DOX. It was previously

24



reported that the simultaneous treatment of cells with free NO and DOX was found not to be effective in inducing a chemosensitizing effect.45 This means that the pretreatment of NO prior to the addition of anticancer agents is crucial in the expression of chemosensitizing effect.23 We pretreated the cells with GSNOHANPs of 600 μg/mL, a concentration range of negligible cytotoxicity (Figure S10 in Supporting information). For the range of DOX concentrations used in our experiments, the GSNO-HANPs promoted therapeutic DOX activities in the range 3.9~75 %. These results indicate that NO generated intracellularly by the GSNOHANPs greatly made the MCF-7 cells vulnerable to DOX treatment, thereby improving DOX efficacy.

% Cell Viability ( Relative to Control)

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

Page 26 of 46

* P< 0.05 *

100

*

*

*

*

DOX HANPs + DOX GSNO-HANPs + DOX

*

*

* *

75

*

*

*

50 * *

25

0 0.01

0.025

0.05

0.1

0.25

0.5

1

DOX Concentration (g/mL)

Figure 4. In vitro chemosenstizing effect of GSNO-HANPs with DOX (n=3). 25


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3.7. Apoptotic activity. Flow cytometry analysis was used for estimation of apoptotic activity of the DOX-combined GSNO-HANPs. The cells were treated with DOX after pretreatment with GSNO-HANPs (300 or 600 µg/mL). Staining of FITC-labeled Annexin V, which identifies early-stage apoptosis, and propidium iodide (PI), which indicates cell permeability and the late-stage apoptosis, was analyzed in MCF-7. The % sum of early/late-stage apoptotic cells was used to evaluate apoptotic activity of each sample. As shown in Figure 5a and b, apoptosis (7.85 %) by the control group is negligible. For the GSNO-HANP-treated group, the proportion of apoptotic cells was 8.94 % and 7.50 % at concentrations of 300 or 600 µg/mL, respectively; neither were significantly different from the control group. This result shows that the GSNO-HANP itself, under our experimental condition, did not exert any significant apoptotic effect on MCF-7 cells. It is likely that the NO released intracellularly by the GSNO-HANPs is an insufficient level to induce apoptosis. In the case of the DOX-treated group, an increased proportion of apoptotic cells was found (14.69 %). Apoptosis in these cells was probably induced by mitochondrial DNA damage by DOX-induced formation of reactive H2O2 and O2•-.47

Interestingly, the MCF-7 cells with pretreated with the GSNO-HANPs and then treated with DOX exhibited significantly increased levels of apoptosis. More 26



apoptosis was induced when the concentration of the GSNO-HANPs increased from 300 (36.15 %) to 600 µg/mL (57.11 %) (Figure 5a and b). This reflects that the enhanced formation of ONOO- by combination of NO and DOX-induced O2•may contribute to an increased apoptotic activity of DOX (Figure 1c). Figure 5c shows FITC-Annexin V staining. Negligible green fluorescence was found in the control group (Figure 5c and d). For cells incubated with only GSNO-HANPs also showed weak green fluorescence. This indicates that there were few apoptotic cells in control samples and the GSNO-HANPs-treated group. In contrast, the MCF-7 cells pretreated with the GSNO-HANPs and then DOX showed obvious blebbing of FITC-Annexin V stained cellular membranes, typically observed in apoptotic cells, and this phenomenon was more apparent in this treatment group than in other groups. These results confirm that GSNO-HANPs lack apoptotic potential on their own but can intensify the pro-apoptotic activity of DOX toward cancer cells.

27


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(a)

GSNO-HANPs (300 g/mL)

Control


0.80 %

0.94 %

0.92 %

7.05 %

8.00 %

6.58 %

GSNO-HANPs (300 g/mL) + DOX

DOX


6.61 %

21.74 %

12.41 %

29.54 %

35.37 %

PI

2.28 %

FITC-Annexin V

(c)

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Control

DOX



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*

Apoptotic Cells (%)

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DOX

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*

*P < 0.05

*

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Intensity (a.u.)

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


*

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10 8 6 4 2 0 control

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DOX

GSNO-HANPs GSNO-HANPs (300 μg/mL) (600 μg/mL) + DOX + DOX

28




Figure 5. (a) The MCF-7 cells estimated by flow cytometry in various sample groups. Q1: cells of necrosis, Q2: cells of late-stage apoptosis, Q4: cells of earlystage apoptosis, and Q3: living cells. The apoptotic activity was quantified by sum of % cells in Q4 and Q2, (b) % of apoptotic cells (% sum of early/late-stage apoptotic cells) (n=3), (c) CLSM analysis of the cells treated by various samples and subsequent Annexin V-FITC staining, and (d) fluorescence intensities of Annexin V-FITC in various sample-treated MCF-7 cells (n=3).

3.8. Quantification of GSNO-HANP-induced intracellular peroxynitrite generation. To support the postulated mechanism for the chemosensitizing effect of NO, DAX-J2 PON Green that has a high selectivity toward ONOO- was adopted for real-time quantitative assay of ONOO- production within the cells.48 As shown in Figure 6, for DOX-treated MCF-7 cells, we found slightly increased ONOOformation probably due to the reaction between DOX semiquinone radicals and the endogenous intracellular NO molecules. GSNO-HANPs also increased the ONOOlevel, which can be ascribed to the reaction released NO and the endogenous intracellular O2•-. It is noted that, compared with the free DOX-treated cells, the MCF-7 cells with sequential treatment of GSNO-HANPs and DOX exhibited increased levels of ONOOformation. This indicates that NO released from the GSNO-HANPs led to an increase in ONOO- levels through the reaction with O2•-, the level of which was elevated by DOX

29


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semiquinone radicals. From the assay of intracellular ONOO-, we could obtain an important information that, although GSNO-HANPs generated the higher level of ONOO- than DOX alone, they neither exhibited the apoptotic activity nor cellular toxicity (Figure S8 in Supporting Information). This indicates that cell damage by GSNO-HANP-induced ONOO- did not reach a level that can induce substantial cell toxicity or apoptosis. Thus, it is confirmed that the major role of GSNO-HANPs is to make MCF-7 cells more susceptible to DOX treatments.

* P < 0.05

0.24

Intensity (a.u.)

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


*

*

*

0.21

*

0.18

0.15

0.12

control

DOX

GSNO-HANPs GSNO-HANPs GSNO-HANPs (300 g/mL) (300 g/mL) (600 g/mL) + DOX + DOX

Figure 6. Intracellular real-time assay of ONOO- using DAX-J2 PON Green for various sample groups (n=3).

3.9. In vivo biodistribution and accumulation of GSNO-HANPs in tumors. To examine the biodistribution and accumulation of GSNO-HANPs 30



within the tumors, we carried out NIRF imaging analyses using Cy5.5-labeled GSNO-HANPs. The NIRF of GSNO-HANPs-Cy5.5 were clearly located in tumor sites (at 1 h). The signals increased to around 4.1 times the initial fluorescence intensity at 9 h, and maintained strong signals up to 1.8 times the initial intensity at 48 h (Figure 7a and b). This indicated the prolonged retention time of GSNOHANPs-Cy5.5 within the tumor site. At 48 h, distribution in major organs and accumulation in tumors was analyzed by observing ex vivo NIRF imaging (Figure 7c and d). These results demonstrated that GSNO-HANPs-Cy5.5 possessed significantly increased systemic circulation and tumor accumulation. Although a certain portion of GSNO-HANPs-Cy5.5 accumulated within the major organs due to the reticuloendothelial system (RES) of each organ, the level of fluorescence signals in the tumor was also significant due to a synergy of CD44-mediated endocytosis and the EPR effect. Furthermore, a distinct distribution of GSNOHANPs-Cy5.5 in the sectioned tumor tissues was detected (Figure 7e), consistent with in vivo biodistribution images. 3.10. In vivo chemosensitizing chemotherapeutic efficacy of GSNOHANPs. The in vivo chemosensitizing effect of GSNO-HANPs to DOX was evaluated. As shown in Figure 8a and b, mice treated with GSNO-HANPs + DOX showed significantly suppressed tumor growth and improved survival rate.

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However, as in the case of PBS and GSNO-HANP, the mice did not show substantial tumor suppression effects, and the tumor rapidly reached up to 2,000 mm3. Interestingly, free DOX moderately suppressed tumor growth until 10 days, but tumor volume increase was certainly accelerated and reached 1,585 mm3 on day 16, suggesting that free DOX activity was an insufficient level to suppress tumor growth. Specifically, the treatment with GSNO-HANPs in combination with DOX inhibited tumor growth effectively, associated with a smaller tumor volume (123 mm3) than free DOX and GSNO-HANP treatment alone on day 14 (985 mm3 and 1,790 mm3, respectively). These results indicated that alone, neither DOX nor GSNO-HANPs could produce proper therapeutic efficacy, whereas pretreatment with GSNO-HANPs resulted in a significant improvement of antitumor efficacy of DOX for solid MCF-7 tumors. Given the significant in vivo antitumor activity of combination treatment with GSNO-HANPs, we examined the potential adverse effects of GSNO-HANPs. Mice treated with PBS, free DOX, GSNO-HANPs, or GSNO-HANPs + DOX did not show significant changes in body weight, which indicated a lack of treatmentinduced toxicity and that the regimes were all well-tolerated in mice (Figure 8c). In the TUNEL assay, significantly greater apoptotic areas were observed in the dissected tumor tissues treated with GSNO-HANPs + DOX compared with tumor

32



tissues from all other groups (Figure 8d). In addition, the corresponding apoptosis rate of the TUNEL assay also supports the pronounced apoptotic activity of GSNOHANPs + DOX (Figure 8e). For further evaluation of systemic toxicity, histological analysis was performed on the dissected major organs and tumor tissues. No groups exhibited any pathological changes in H&E staining of the major organs, suggesting no significant systemic toxicity of multiple dosages (Figure 9). These in vivo results show that GSNO-HANPs were able to significantly accumulate in solid MCF-7 tumors and effectively suppress the tumor growth when combined with DOX treatment.

(a)

(b)

Cy5.5

GSNO-HANPs -Cy5.5

(e) (c)

(d)

Cy5.5

GSNO-HANPs -Cy5.5

Cy5.5

Fluorescent tumor slice images

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

Page 34 of 46

GSNO-HANPs-Cy5.5

Figure 7. Evaluation of biodistributions of Cy5.5-labeled GSNO-HANPs in tumor-

33


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bearing mice. (a) monitoring of in vivo NIRF signals after i.v. administration, (b) NIRF intensity of tumor tissues, (c) ex vivo NIRF images, and (d) quantification of dissected organs and tumors, and (e) fluorescence tumor tissue analysis 48 h postinjection stained with DAPI and observed under CLSM.

Tumor volume (mm3)

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PB

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S

Days

s

NP HA O-

N GS

Ps AN -H X NO + DO S G

X

Fr

ee

DO

Figure 8. In vivo chemosensitizing effect of GSNO-HANPs with DOX. (a) antitumor efficacies, (b) survival rate, (c) changes in body weights, (d) TUNEL immunofluorescent staining of dissected tumors stained with DAPI and observed under CLSM, and (e) the apoptosis rate of TUNEL assay. 34



Figure 9. Histopathologic evaluation of systemic toxicities in the major organs.

4. CONCLUSIONS

In this study, self-assembled GSNO-incorporated HANPs were successfully developed. The assembled structure of GSNO-incorporated HANPs greatly improved the aqueous stability of GSNO and allowed efficient delivery of NO into the MCF-7 cancer cells. In vitro experiments showed that effective NO

35


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generation within the cancer cells augmented the therapeutic activity of DOX through the sensitization of the MCF-7 cells to DOX. GSNO-HANP also improved the apoptotic activity of DOX toward MCF-7 cells. In vivo experiments showed that combination treatment with GSNO-HANPs and DOX effectively suppressed tumor growth. GSNO-HANPs may be of benefit in DOX therapy in that they can lower the dose of DOX treatment necessary to achieve the anticancer efficacy possible with a relatively higher dose of DOX. This may result in the fewer adverse effects of DOX treatments. Taken together, our findings show that intracellular NO-generating HANPs may serve significant roles in sensitizing many classes of cancers for chemotherapy.

Supporting Information Characterization data of the GSNO-PLGA-HA (1H NMR, FT-IR, UV-Vis spectra), pyrene fluorescence data for CMC measurement, the TEM image and the distribution of the GSNO-HANP size by DLS, CLSM images for cellular uptake of FITC-labeled GSNO-HANPs and Cy5.5-labeled GSNO-HANPs, in vitro toxicity of GSNO-HANPs, and time-dependent monitoring of intracellular NO generation. E-mail Address of Corresponding Authors 36



[email protected] & [email protected] ORCID Sang Cheon Lee: 0000-0002-8560-0173 Hong Jae Lee: 0000-0003-3650-6436 Kyung Hyun Min : 0000-0002-6705-5028 Da Eun Kim : 0000-0002-3693-6383 Kiyuk Chang: 0000-0003-3456-8705 Chan Woo Kim: 0000-0001-8430-3903 Author Contributions ‡These authors contributed equally to this paper.

ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) & funded by the Korean government (MSIP&MOHW) (No. 2017M3A9E4048170) and Basic Science Research Program (No. 2014R1A2A1A10051669) of the National Research Foundation of Korea (NRF).

REFERENCES (1) Carpenter, A. W.; Schoenfisch, M. H. Nitric Oxide Release: Part II. Therapeutic Applications. Chem. Soc. Rev. 2012, 41, 3742-3752. (2) Mocellin, S.; Bronte, V.; Nitti, D. Nitric Oxide, a Double Edged Sword in Cancer Biology: Searching for Therapeutic Opportunities. Med. Res. Rev. 2007, 27, 37


Page 38 of 46

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317-352. (3) Wimalawansa, S. J. Nitric Oxide: New Evidence for Novel Therapeutic Indications. Expert Opin. Pharmacother. 2008, 9, 1935-1954. (4) Kiziltepe, T.; Hideshima, T.; Ishitsuka, K.; Ocio, E. M.; Raje, N.; Catley, L.; Li, C. Q.; Trudel, L. J.; Yasui, H.; Vallet, S.; Kutok, J. L.; Chauhan, D.; Mitsiades, C. S.; Saavedra, J. E.; Wogan, G. N.; Keefer, L. K.; Shami, P. J.; Anderson, K. C. JSK, a GST-Activated Nitric Oxide Generator, Induces DNA Double-Strand Breaks, Activates DNA Damage Response Pathways, and Induces Apoptosis In Vitro and In Vivo in Human Multiple Myeloma Cells. Blood 2007, 110, 709-718. (5) Huerta, S.; Chilka, S.; Bonavida, B. Nitric Oxide Donors: Novel Cancer Therapeutics (Review). Int. J. Oncol. 2008, 33, 909-927. (6) Brune, B.; von Knethen, A.; Sandau, K. B. Nitric Oxide (NO): an Effector of Apoptosis. Cell Death Differ. 1996, 6, 969-975. (7) Choudhari, S. K.; Chaudhary, M.; Bagde, S.; Gadbail, A. R.; Joshi, V. Nitric Oxide and Cancer: a Review. World J. Surg. Oncol. 2013, 11, 118. (8) Tan, L.; Huang, R.; Li, X.; Liu, S.; Shen, Y. M. Controllable Release of Nitric Oxide and Doxorubicin from Engineered Nanospheres for Synergistic Tumor Therapy. Acta Biomaterialia 2017, 57, 498-510. (9) Fan, J. He, Q.; Liu, Y.; Zhang, F.; Yang, X.; Wang, Z.; Lu, N.; Fan, W.; Lin,

38



Page 40 of 46

L.; Niu, G.; He, N.; Song, J.; Chen, X. Light-Responsive Biodegradable Nanomedicine

Overcomes

Multidrug

Resistance

via

NO-Enhanced

Chemosensitization. ACS Appl. Mater. Interfaces 2016, 8, 13804-13811. (10) Munaweera, I.; Shi, Y.; Koneru, B.; Patel, A.; Dang, M. H.; Di Pasqua, A. J.; Balkus Jr., K. J. Nitric Oxide- and Cisplatin-Releasing Silica Nanoparticles for Use against Non-Small Cell Lung Cancer. J. Inorganic Biochem. 2015, 153, 23-31. (11) Csonka, C.; Páli, T.; Bencsik, P.; Görbe1, A.; Ferdinandy, P,; Csont, T. Measurement of NO in Biological Samples. British J. Pharmacol. 2015, 172, 16201632. (12) Nichols, S. P.; Storm, W. L.; Koh, A.; Schoenfisch, M. H. Local Delivery of Nitric Oxide: Targeted Delivery of Therapeutics to Bone and Connective Tissues. Adv. Drug Deliv. Rev. 2012, 64, 1177-1188. (13) Broniowska, K. A.; Hogg, N. The Chemical Biology of S-Nitrosothiols. Antioxidants & Redox Signaling 2012, 17, 969-980. (14) Nakagawa, H. Photocontrollable NO-Releasing Compounds and Their Biological Applications. J. Clin. Biochem. Nutr. 2016, 58, 2-6. (15) Huh, D. N.; Czer, E. T.; Cordova, K. E.; Chow, W. I.; Moore, C. E.; Rheingold, A. L.; Daley, C. J. Metal Nitrosyl Chemistry: Interesting Oxidation and Nitrosylation of a Metal-Bound Ligand Framework in a Diamido-bis (phosphine)

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Ruthenium (II) Complex. Inorganica Chimica Acta 2016, 450, 236-242. (16) Lu, Y.; Slomberg, D. L.; Schoenfisch, M. H. Nitric Oxide-Releasing Chitosan Oligosaccharides as Antibacterial Agents. Biomaterials 2014, 35, 1716-1724. (17) Huang, Z.; Fu, J.; Zhang, Y. Nitric Oxide Donor-Based Cancer Therapy: Advances and Prospects. J. Med. Chem. 2017, 60, 7617-7635. (18) Shami, P. J.; Saavedra, J. E.; Wang, L. Y.; Bonifant, C. L.; Diwan, B. A.; Singh, S. V.; Gu, Y.; Fox, S. D.; Buzard, G. S.; Citro, M. L.; Waterhouse, D. J.; Davies, K. M.; Ji, X.; Keefer, L. K. JS-K, a Glutathione/Glutathione S-Transferase-Activated Nitric Oxide Donor of the Diazeniumdiolate Class with Potent Antineoplastic Activity. Mol. Cancer Ther. 2003, 2, 409-417. (19) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Thacher, T. N.; Antonijevic, S.; Cavadini, S.; Demurtas, D.; Stergiopulos, N.; Hubbell, J. A. Micelles for Delivery of Nitric Oxide. J. Am. Chem. Soc. 2009, 131, 14413-11418. (20) Bradley, S. A.; Steinert, J. R. Characterisation and Comparison of Temporal Release Profiles of Nitric Oxide Generating Donors. J. Neuroscience Methods 2015, 245, 116-124. (21) Broniowska, K. A.; Hogg, N. The Chemical Biology of S-Nitrosothiols. Antioxidants & Redox Signaling 2012, 17, 969-980. (22) Seabra, A. B.; de Lima, R.; Calderón, M. Nitric Oxide Releasing

40



Nanomaterials for Cancer Treatment: Current Status and Perspectives, Curr. Top. Med. Chem. 2015, 15, 298-308. (23) Duong, H. T. T.; Kamarudin, Z. M.; Erlich, R. B.; Li, Y.; Jones, M. W.; Kavallaris, M.; Boyer, C.; Davis, T. P. Intracellular Nitric Oxide Delivery from Stable NO-Polymeric Nanoparticle Carriers. Chem. Commun. 2013, 49, 4190-4192. (24) Lee, H. J.; Kim, D. E.; Park, D. J.; Choi, G. H.; Yang, D. N.; Heo, J. S.; Lee, S. C. pH-Responsive Mineralized Nanoparticles as Stable Nanocarriers for Intracellular Nitric Oxide Delivery. Colloids and Surfaces B: Biointerfaces 2016, 146, 1-8. (25) Lee, H.; Ahn, C. H.; Park, T. G. Poly[lactic-co-(glycolic acid)]-Grafted Hyaluronic Acid Copolymer Micelle Nanoparticles for Target-Specific Delivery of Doxorubicin. Macromol. Biosci. 2009, 9, 336-342. (26) Wilhelm, M.; Zhao, C.; Wang, Y.; Xu, R.; Winnik, M. A.; Mura, J.; Riess, G.; Croucher, M. D. Poly(styrene-ethylene oxide) Block Copolymer Micelle Formation in Water: A Fluorescence Probe Study. Macromolecules 1991, 24, 10331040. (27) Kim, S.; Kwag, D. S.; Lee, D. J.; Lee, E. S. Acidic pH-Stimulated Tiotropium Release from Porous Poly(lactic-co-glycolic acid) Microparticles Containing 3Diethylaminopropyl-Conjugated Hyaluronate. Macromol. Res. 2016, 24, 176-181.

41


Page 42 of 46

Page 43 of 46 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


(28) Jegal, J. H.; Choi, G. H.; Lee, H. J.; Kim, K. D.; Lee, S. C. Surface Engineering of Titanium with Simvastatin-Releasing Polymer Nanoparticles for Enhanced Osteogenic Differentiation. Macromol. Res. 2016, 24, 83-89. (29) Choi, K. Y.; Chung, H.; Min, K. H.; Yoon, H. Y.; Kim, K.; Park, J. H.; Kwon, I. C.; Jeong, S. Y. Self-Assembled Hyaluronic Acid Nanoparticles for Active Tumor Targeting. Biomaterials 2010, 31, 106-114. (30) Park J.-W.; Means, G. An Unusually Stable S-Nitrosothiol from Glutathione. Arch. Pharm. Res. 1989, 12, 257-258. (31) Kwon, G: Naito, M.; Yokoyama, M,; Okano, T.: Sakurai, Y.: Kataoka, K. Micelles Based on AB Block Copolymers of Poly(ethylene oxide) and Poly(βbenzyl L-aspartate). Langmuir 1993, 9, 945-949. (32) Wang, B.; Jiang, W.; Yan, H.: Zhang, X.: Yang, L.: Deng, L,: Singh, G. K.: Pan, J. Novel PEG-graft-PLA Nanoparticles with the Potential for Encapsulation and Controlled Release of Hydrophobic and Hydrophilic Medications in Aqueous Medium. Int. J. Nanomedicine 2011, 6, 1443-1451 (33) Lee, R.-S.: Huang, Y.-T. Tuning the Hydrophilic–Hydrophobic Balance of Block-Graft Copolymers by Click Strategy: Synthesis and Characterization of Amphiphilic PCL-b-(PaN3CL-g-PBA) Copolymers. Polymer J. 2010, 42, 304-312. (34) Ito, T.: Sun, L.: Bevan, M. A.: Crooks, R. M. Comparison of Nanoparticle

42



Size and Electrophoretic Mobility Measurements Using a Carbon-Nanotube-Based Coulter Counter, Dynamic Light Scattering, Transmission Electron Microscopy, and Phase Analysis Light Scattering. Langmuir 2004, 20, 6940. (35) Kaasalainen, M.: Aseyev, V.; von Haartman, E.: Karaman, D. S.: Mäkilä, E.: Tenhu, H.: Rosenholm, J.: Salonen, J. Size, Stability, and Porosity of Mesoporous Nanoparticles Characterized with Light Scattering. Nanoscale Research Letters 2017, 12, 74. (36) Shah, S. U.; Socha, M.; Fries, I.; Gibaud, S. Synthesis of S-NitrosoglutathioneAlginate for Prolonged Delivery of Nitric Oxide in Intestines. Drug Deliv. 2016, 23, 2927-2935. (37) Thomas, R. G.; Muthiah, M.; Moon, M.; Park, I.-K.; Jeong, Y. Y. SPION Loaded Poly(L-lysine)/Hyaluronic Acid Micelles as MR Contrast Agent and Gene Delivery Vehicle for Cancer Theranostics. Macromol. Res. 2017, 25, 446-451. (38) Tan, J. X.; Wang, X. Y.; Li, H. Y.; Su, X. L.; Wang, L.; Ran, L.; Zheng, K.; Ren, G. S. HYAL1 Overexpression is Correlated with the Malignant Behavior of Human Breast Cancer. Int. J. Cancer 2011, 128, 1303-1315. (39) Rim, H. P.; Min, K. H.; Lee, H. J.; Jeong, S. Y.; Lee, S. C. pH-Tunable Calcium Phosphate Covered Mesoporous Silica Nanocontainers for Intracellular Controlled Release of Guest Drugs. Angew. Chem. Int. Ed. 2011, 50, 8853-8857.

43


Page 44 of 46

Page 45 of 46 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


(40) Washko, P.; Rotrosen, D.; Levine, M. Ascorbic Acid in Human Neutrophils. Am. J. Clin. Nutr. 1991, 54, 1221S-1227S. (41) Han, H. S.; Thambi, T.; Choi, K. Y.; Son, S.; Ko, H.; Lee, M. C.; Jo, D.-G.; Chae, Y. S.; Kang, Y. M.; Lee, J. Y.; Park, J. H. Bioreducible Shell-Cross-Linked Hyaluronic

Acid

Nanoparticles

for

Tumor-Targeted

Drug

Delivery.

Biomacromolecules 2015, 16, 447-456. (42) Zhou, X.; He, P. Improved Measurements of Intracellular Nitric Oxide in Intact Microvessels using 4,5-Diaminofluorescein Diacetate. Am. J. Physiol. Heart Circ. Physiol. 2011, 301, H108-H114. (43) Kalivendi, S. V.; Kotamraju, S.; Zhao, H.; Joseph, J.; Kalyanaraman, B. Doxorubicin-Induced Apoptosis is Associated with Increased Transcription of Endothelial Nitric-Oxide Synthase. Effect of Antiapoptotic Antioxidants and Calcium. J. Biol. Chem. 2001, 276, 47266-47276. (44) Frederiksen, L. J.; Sullivan, R.; Maxwell, L. R.; Macdonald-Goodfellow, S. K.; Adams, M. A.; Bennett, B. M.; Siemens, D. R.; Graham, C. H. Chemosensitization of Cancer In Vitro and In Vivo by Nitric Oxide Signaling. Clin. Cancer Res. 2007, 13, 2199-2206 (45) Evig, C. B.; Kelley, E. E.; Weydert, C. J.; Chu, Y.; Buettner, G. R.; Burns, C. P. Endogenous Production and Exogenous Exposure to Nitric Oxide Augment

44



Doxorubicin Cytotoxicity for Breast Cancer Cells but not Cardiac Myoblasts. Nitric Oxide 2004, 10, 119-129. (46) Czapski, G.; Goldstein, S. The Role of the Reactions of ·NO with Superoxide and Oxygen in Biological Systems: a Kinetic Approach. Free Radic. Biol. Med. 1995, 19, 785-794. (47) Mizutani, H.; Tada-Oikawa, S.; Hiraku, Y.; Kojima, M.; Kawanishi, S. Mechanism of Apoptosis Induced by Doxorubicin through the Generation of Hydrogen Peroxide. Life Sci. 2005, 76, 1439-1453. (48) Luo, Z.; Zhao, Q.; Liu, J.; Liao, J.; Peng, R.; Xi, Y.; Diwu, Z. Fluorescent Real-Time Quantitative Measurements of Intracellular Peroxynitrite Generation and Inhibition. Anal. Biochem. 2017, 520, 44-48.

45


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Intracellular NO-Releasing Hyaluronic Acid-Based Nanocarriers: A

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