nanogels for co-delivery of doxorubicin and 6‑mercaptopurine

Accepted Manuscript A novel multi stimuli-responsive PEGylated hybrid gold/nanogels for co-delivery of doxorubicin and 6'mercaptopurine

Marjan Ghorbani, Hamed Hamishehkar PII: DOI: Reference:

S0928-4931(17)34643-X doi:10.1016/j.msec.2018.07.019 MSC 8731

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

28 November 2017 7 June 2018 8 July 2018

Please cite this article as: Marjan Ghorbani, Hamed Hamishehkar , A novel multi stimuliresponsive PEGylated hybrid gold/nanogels for co-delivery of doxorubicin and 6'mercaptopurine. Msc (2018), doi:10.1016/j.msec.2018.07.019

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ACCEPTED MANUSCRIPT A novel multi stimuli-responsive PEGylated hybrid gold/nanogels for co-delivery of doxorubicin and 6-mercaptopurine

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Marjan Ghorbani1,2, Hamed Hamishehkar2*

Stem cell research center, Tabriz University of Medical Sciences, Tabriz, Iran.

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Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran.

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*Corresponding author:

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Hamed Hamishehkar: +98 41 33363231; Fax: +98 41 33363231; E-mail addresses:

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[email protected].

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ACCEPTED MANUSCRIPT Abstract The clinical applications of anticancer drugs is restricted due to the incomplete delivery to the cancerous tissue and the numerous drug resistance mechanisms involved in malignant cells. In this regard, stimuli‐responsive nanomaterials offer a promising prospect to deal with these

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concerns. In the present study, ternary responsive hybrid gold/nanogels (Au/NGs) were synthesized as a new nanoplatform to simultaneously carry two anticancer drugs, i.e.,

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doxorubicin (DOX) and 6-mercaptopurine (MP). For this purpose, these drugs were successfully

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loaded (the loading capacity of 23% and 11%, respectively) into the hybrid Au/NGs by electrostatic interaction (DOX) and Au-S bonds (MP). The triggered drug release ability of

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hybrid Au/NGs was assessed by comparing the environments of simulated physiological and

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tumor tissue. The incorporation of disulfide bond linkers, pH sensitive, and thermosensitive polymeric segments endowed the NGs with an excellent property in reducing acidic and

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hyperthermia environments, which greatly facilitates drug release in tumor cells. Intracellular

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tracking of DOX@MP-Au/NGs confirmed efficient accumulation and cellular uptake of developed NGs and the cytotoxicity studies showed a pronounced tumor inhibition compared

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with free DOX@MP. It was concluded that the new ternary-responsive NGs have great potential

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for co-delivery of DOX and MP and can be used in efficient cancer therapy.

Key words: Cancer, Drug delivery; Nanogels; Ternary responsiveness; Doxorubicin; 6Mercaptopurine.

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1. Introduction Today, although chemotherapy is an important pathway in cancer treatment, the administration of anticancer drugs shows nonspecific drug biodistribution in both normal and cancer tissues.

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Therefore, to provide the required drug concentration level in the target site, a large dosage of the

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drug is often used, which leads to the lethal side effects and failure of chemotherapy [1,2]. A

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suitable drug delivery system should provide and hold the drug being transported in the

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bloodstream and only release it in the tumor tissue. For this purpose, nanotechnology-based therapeutics have been considered as one of the potential solutions for cancer chemotherapy

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because of increased drug accumulation in the solid tumor via nano-carriers by the enhanced

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permeability and retention (EPR) effect [3–5]. Among various nanoparticles (NPs) introduced, three-dimensional polymeric networks, which are presented as nanogels (NGs), have shown

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appealing potentials in the area of drug delivery. These gels have many advantages including a)

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nanoscale size, b) flexibility and robustness properties, c) superior colloidal stability, d) high water swelling capacity, e) fine control over particle size, and f) smart responsiveness to

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external stimuli such as temperature, pH, and redox triggers [6,7]. Conjugating polyethylene

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glycol (PEG) onto the surface of NGs (PEGylation) would be an ideal approach to avoid recognition and clearance of the NPs by the reticuloendothelial system (RES), which increases the circulation half-life of NPs [8,9]. However, the hydrophilic shielding layer of PEG may provide a negative effect on the cellular uptake and intracellular distribution due to the steric hindrance. Besides, it may act as a diffusion barrier and cause incomplete release of the encapsulated drugs [10]. To overcome this problem, removing PEG layer from the outer-shell of nanocarrier in the cancerous tissue is an ideal strategy [11,12]. Previously published reports have

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ACCEPTED MANUSCRIPT highlighted the positive role of redoxresponsive carriers to improve the therapeutic efficacy of drug delivery systems because of their degradability in redox condition [13–15]. Through this path, the purpose is to achieve a readily controlled drug release upon arrival of NGs at the target site. To address this issue, different characteristics of tumor tissue from normal ones such as a

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lower pH, higher temperature, and a higher level of reducing agent, i.e., glutathione (GSH), may

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be used. Therefore, stimuli-responsive nanocarriers, especially thermo- [16], pH- [17,18] and

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redox- [19,20] and multi-responsive [21,22] nano-vehicles have received extra attention in cancer drug delivery systems for facilitating the smart drug release. Moreover, gold (Au) NPs

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have gained increasing interests in medicine and biology because of their unique surface

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chemical, physical properties for transporting, loading/unloading drugs through the Au-S bond stable in physiological conditions [22–24], and improving the anticancer effects of drugs [23,24].

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Therefore, Au-based NPs can be considered because of many advantages of Au: (1) small

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diameters that assist tumor penetration upon systemic delivery, (2) biocompatibility, (3) easily bioconjugation with drugs and imaging agents through the Au−S bond for the attachment of

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desired molecules and (4) efficiently conversion of light‐to‐heat applicable in photo-thermal

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therapy. Theses potentials led to recently application of Au NPs in combination with other therapies, such as chemotherapy, gene regulation, and immunotherapy, for enhanced anti‐tumor

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effects [25]. The toxicity of Au NPs limits its direct application in drug delivery [26,27] which can be overcome by covering the surface of NPs by incorporation of biocompatible polymers. In this connection, simultaneous delivery of multiple therapeutic agents, named as combination therapy strategy, introduces several merits: a) minimizing the amount of dose usage for each drug and consequently reducing dose-dependent side effects, b) improving the therapeutic effects, and c) overcome the multidrug resistance [28]. Thus, the design of a delivery system

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ACCEPTED MANUSCRIPT with all the features described above i.e. being a) nano sized, b) composed of Au, c) PEGylated during blood distribution and un-PEGylated after accumulation in cancerous tissue, d) pH, thermo, and redox responsive, and finally e) loaded with more than one cytotoxic drug is extremely important and exciting. Herein, we developed a new type of PEGylated smart hybrid

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Au/NGs to construct the co-delivery system of two anticancer drugs (DOX and MP) for the

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combination cancer therapy. The successful development of our proposed stealth NGs in co-

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delivery of two anti-cancer agents to the tumor tissue and drugs accumulation over there

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(Scheme 1), may open new horizons for the efficient cancer therapy.

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Scheme 1. Schematic illustration of the advantages and capabilities of temperature/pH/reductionresponsive nanogels improve cancer therapy after accumulation in cancerous tissue via enhanced permeability effect.

2. Experimental section

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ACCEPTED MANUSCRIPT 2.1. Materials N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), N,N,Nʹ,Nʹʹ,Nʹʹpentamethyldiethylenetriamine

(PMDETA),

(N,Ndimethylamino)ethyl

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(DMAEMA), 2-bromoisobutyrylbromide (BIBB), potassium persulfate (KPS) and maleic

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anhydrid (MAn) were purchased from Merck Chemicals. Itaconic acid (IA), Hydrogen

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tetrachloroaurate (ш) trihydrate (HAuCl4), N,N’-bis(acryloyl)cystamine (BAC), gluthation

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(GSH), 6-mercaptopurine (MP), sodium borohydride (NaBH4), Copper (I) bromide (CuBr) and

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methoxy poly(ethylene glycol) (mPEG-OH, 5000 Da) were purchased from Sigma-Aldrich. DOX was donated by Exir Nano Sina Company (Iran). DMF was totally dried and then distilled

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under reduced pressure. Tetrahydrofuran (THF) was dried and distilled after refluxing for 3 h. 3-

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(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other biological reagents

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were obtained from Gibco (Carlsbad, USA). 2.2. Synthesis of macroinitiator

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Triethylamine (TEA, 6 mL, 48 mmol) and mPEG 5000 (5 g, 2.5 mmol) were mixed in 55 mL of

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anhydrous THF and was cooled in an ice-water bath for 1h. Then, BIBB (2.75 mL, 21 mmol) was dissolved in 25 mL of THF and added to the solution in a drop-wise manner and stirred for

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48 h. The resulting solution was filtered and the solvent was vaporized under reduced pressure (Edwards RV3 vacuum pump, UK). After that, the product was dissolved in water and extracted five times with dichloromethane. After separating the two phases, the organic phase was washed with 1 M HCl, 1 M NaOH, and saturated aqueous NaCl solution and then, dried over magnesium sulfate for 24 h. The precipitate was obtained in a large excess of ice-cold diethyl ether and dried in a vacuum oven (VO400, Memmert, Germany) for 24 h.

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ACCEPTED MANUSCRIPT 2.3. Synthesis of block copolymer mPEG-b-P(NIPAAm-co-HEMA) (PEG-copolymer) By using an atom transfer radical polymerization method, the block copolymer mPEG-bP(DMAEMA-co-HEMA) was synthesized in a 100 mL three-necked flask under nitrogen inlet and a magnet stirrer. 4 g mPEG-Br (0.168 mmol) and 60 mg CuBr (0.35 mmol) were added to a

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flask, and purged with N2 to remove oxygen by three cycles. After 30 min, deoxygenated

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cyclohexanone (30 mL) was added and stirred for further time. Next, deoxygenated ligand of PMDETA (80 µL, 0.35 mmol) was added to the solution and the change of color was happened

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from cloudy to clear green. Then, deoxygenated HEMA (3.8 g, 30 mmol) and NIPAAm (4 g, 35 mmol) were added to the solution and the flask was immersed in an oil-bath by heating at 70 °C

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under nitrogen atmosphere for 24 h. To quench the reaction, the solution was diluted with 30 mL

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of acetone and put into a dialysis bag (molecular weight cut off 1000 Da, Sigma-Aldrich, USA). After removing the unreacted monomer and catalyst, the polymer was obtained by freeze-drying

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method (FD-10, Pishtaz Engineering, Iran). According to the 1HNMR data, the molecular weight

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(Mn) of synthesized copolymer was calculated by the comparison of the peaks integration at 3.9

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ppm of HEMA, 3.76 ppm of NIPAAm and a reference peak at 3.65 ppm of mPEG. 2.4. Synthesis of PEG-copolymer-g-maleic acid (PEG-copolymer-g-MAc)

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The PEG-copolymer-g-MAc was prepared to provide the double bonds as crosslinker agents in the backbone of copolymer. The PEG-copolymer-g-MAc was prepared according to the reported procedure with minor modification [29,30]. Briefly, 1g of synthesized PEG-copolymer was added into 15 mL of dichloromethane and after complete dissolvation, the MAn (600 mg, 6 mmol) was added to the solution under nitrogen atmosphere. After 24h, the soluble products

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ACCEPTED MANUSCRIPT were purified by dialysis bag to remove the excess MAn monomers and then was lyophilized for further experiments. 2.5. Preparation of ternary smart PEG-b-[P(NIPAAM-co-HEMA-g-MAc)-BAC-co-

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DMAEMA] NGs

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The radical polymerization procedure was carried out as follows [31]: BAC (40 mg, 0.16 mmol),

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DMAEMA (50 mg, 0.5 mmol), PEG-copolymer-g-MAc (100 mg, 0.76 mmol), and AIBN (2.65 mg, 0.016 mmol) were dissolved in 10 mL DMF and equipped with a magnetic stir bar under

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nitrogen atmosphere. After the reaction flask was purged with N2 for 25 min, the flask was put

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into a preheated heating oil bath at 70 °C. The polymerization was stopped by cooling in an ice water bath and exposure to air after 75 min. Finally, the solution was diluted with 30 mL of

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water and introduced into a dialysis bag (molecular weight cut off 1000 Da) to remove the

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unreacted monomer and solvent and dialyzed during 3 day to achieve NGs.

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2.6. In situ preparation of hybrid Au/NGs Hybrid Au/NGs was synthesized by in situ entrapping of Au NPs in the matrix of NGs [32].

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HAuCl4 aqueous solution (100 µL, 50 mM) was added into 10 mL NGs (5 mg.mL-1) and

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vigorously stirred in an ice bath for 2 min and this mixture quickly became a yellow transparent dispersion. Then, the reduction of the entrapped Au3+ ions was carried out with freshly prepared NaBH4 aqueous solution (10 mM in 0.3 M NaOH). The mixture was stirred in an ice bath for several minutes and the pale yellow solution quickly convert to the characteristic red color during the reduction of Au3+ ions. Freshly prepared NaBH4 aqueous solution was slowly dropped into the dispersiono. Since the mol amount of NaBH4 is low and it is immediately used to reduce the gold salt, therefor it seems that the reducing of the disulfide bonds occurs at a lower rate. The 9

ACCEPTED MANUSCRIPT stability assessment of developed nanocarrier by TEM and PCS experiments will be vital to check the integrity of system after addition of NaBH4. The solution hybrid Au/NGs was transferred into the dialysis bag with a molecular weight cut off of 3500 Da and the solution was extensively dialyzed against PBS before drug loading experiments.

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2.7. Characterization

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Fourier transform infrared (FTIR) spectra were obtained in the range of 400–4000 cm−1 on a Tensor 27 spectrometer (Bruker, Germany). 1H-NMR spectra were verified at 25 °C in CDCl3 on

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a 400 MHz NMR spectrometer (Bruker, Germany). The hydrodynamic size, polydispersity index

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(PDI) and zeta potential of the NGs dispersed in water were recorded on a zeta-sizer (ZetasizerZS, Malvern, UK). The morphologies of blank and hybrid NGs were determined by a

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transmission electron microscopy instrument (CM120, Philips, Germany) with an accelerating

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voltage of 200 kV. For determination of the lower critical solution (LCST) parameter, the temperature of the solution was set from 25 to 50 °C at wavelength of 600 nm and the spectra

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were recorded by UV-visible spectrophotometer (Cary-100 UV-Vis, USA). The stability of

during 3 days.

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hybrid Au/NGs dispersion (1 mg.ml-1 in water) was assessed by UV-vis spectrophotometer

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2.8. DOX and MP loading in the hybrid Au/NGs (DOX@MP-Au/NGs) MP and DOX were chosen as anticancer drugs model and loaded by the coupling reaction (bonding -S groups of MP to the surface of Au NPs) and electrostatic conjugation between positively charged amino groups of DOX.HCl with negatively charged MAc in the hybrid Au/NGs. The solution of MP (2 mg in 2.5 mL DMSO) was mixed to Au/NGs (1 mL, 5 mg mL-1) suspension in dropwise manner under nitrogen atmosphere for 30 min and stirred for 24 h. Then, 10

ACCEPTED MANUSCRIPT to purify the MP-Au/NGs, the suspension was dialyzed against PBS for 2 days to eliminate DMSO solvent and unreacted MP. Moreover, 500 µL DOX solution (10 mg.mL-1) in PBS was added to MP-Au/NGs (1 mL, 10 mg.mL-1) suspension to shake for 24 h at room temperature, then, the purification of DOX@MP-Au/NGs from the unloaded drug was performed by using the

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Amicon® filter (molecular weight cutoff 100 kDa, Millipore, UK). The unloaded drug

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concentrations were analyzed by UV-vis spectroscopy at 480 nm and 310 nm for DOX and MP,

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respectively. Both drugs showed sharp peaks at the specified wavelength without any interfere to

calculated according to the following equations:

Mass of drug in NGs 100 Mass of NGs

EE (%,w/w) =

Mass of drug in NGs ×100 Mass of initial added drug

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LC  %, w/w  

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each other. The encapsulation efficiency (EE) and loading capacity (LC) percentages were

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The unloaded drugs were removed by Amicon® filters and then the NGs were used for the rest of

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studies.

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2.9. In-vitro release studies of DOX and MP To investigate the potential ability of NGs to smart release of loaded drugs, the responsiveness behavior of DOX@MP-Au/NGs was studied in different simulated cancerous tissue mediums and physiological condition. The release profiles of DOX and MP was assessed in phosphate buffer (pH 7.4) with different concentrations of GSH (2 µM and 10 mM) under normothermia (37 °C) and hyperthermia (41 °C). To carefully evaluate the temperature, pH and redoxdependent drug release, the release medium of drugs was adjusted in phosphate buffer (pH 6) 11

ACCEPTED MANUSCRIPT with simulated normal (37 °C, GSH 2 µM) and cancerous tissue conditions (41 °C, GSH 10 mM). DOX@MP-Au/NGs suspension (1 mL, 5 mg.mL-1) was transferred into dialysis bag (molecular weight cut off 12 KDa, Sigma-Aldrich, USA) and dialyzed against mentioned conditions in the volume of 25 mL. At desired time intervals, the specified amounts of the

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solution were removed from the reservoir and the same volume of fresh medium was added to

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the reservoir. To determine the amounts of released drugs, the concentrations of drugs were

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evaluated using UV-vis spectroscopy.

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2.10. Hemocompatibility test

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First, human blood was prepared from healthy donors and centrifuged at 1000 rpm for 10 min to separate the plasma. Then, the red blood cells (RBC) were centrifuged three times with cold PBS

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at 1000 rpm for 10 min. To obtain a concentration of 10 w/v% RBC suspension (0.1 ml), the

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pellet was resuspended in PBS and then added to 0.9 mL of different concentrations of NGs (500 to 2000 µg.ml-1) and kept for 60 min at 37°C. Moreover, distilled water and saline solution was

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considered as positive control (100% lysis) and negative control (0 % lysis), respectively. The

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absorbance of supernatant was recorded after centrifugation (2000 rpm for 5 min) by UV-vis

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spectroscopy at 540 nm. The hemolysis ratio was calculated by the following formula:

Hemolysis Rate % =

Dtest  Dnegative control

Dpositive control  Dnegative control

100

2.11. in-vitro biocompatibility and cell cytotoxicity studies To assess the therapeutic ability of DOX@MP-Au/NGs, MTT cell assay was evaluated against MCF-7 cell line (National Cell Bank of Iran, Pasteur Institute) for measuring the cell viability and the results were compared with the DOX, MP, the mixture of MP@DOX and blank NGs. 12

ACCEPTED MANUSCRIPT The cells were cultured in RPMI 1640 medium (GIBCO Invitrogen GmbH, Germany) with 10% fetal bovine serum (heat-inactivated, Gibco), 100 IU.mL-1 penicillin and 100 µg.mL-1 streptomycin and incubated at 37 °C in 5% CO2 and 95% air with more than 95% humidity. After 24 h, the cells were seeded into a 96-well plate at a density of 1×104 cells/well. After an

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overnight, the culture medium was removed and the cells were treated with various

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concentrations of the DOX, MP, and the mixture of MP@DOX, MP@DOX-Au/ NGs and blank

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NGs. After 24 and 48 h, the medium was removed and the MTT solution (5 mg.mL-1) was added to incubate for further 4 h. The optical density (OD) at a wavelength of 570 nm was recorded

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using a microplate reader (Elx808, Biotek, USA) and the results were obtained by comparing

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with the control cells.

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2.12. In-vitro cell uptake

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The cellular uptake of DOX@MP-Au/NGs was estimated against MCF-7 cell line by flow cytometry analysis. The MCF-7 cells were seeded in 6-well plates at 3 × 105 cells /well and after

DOX@MP) and DOX@MP for 3h. Thereafter, the cells were washed three times with PBS and

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incubation for 24h, the cells were incubated with DOX@MP and DOX@MP-Au/NGs (1 µg.mL-

the cell uptake was measured using a FACS calibur flow cytometer based on the DOX

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fluorescence (Becton Dickinson Immunocytometry Systems, San Jose, CA,USA). 2.13. In-vitro cell apoptosis 2.13.1. Cell cycle arrest The cells were seeded at a density of 4 × 105 cells/mL in a six well plate and after incubation for 24 h, the cells were treated with Au/NGs, DOX@MP and DOX@MP-Au/NGs. Then, the cells were harvested and centrifuged at 300×g for 10 min at 4 °C. The pellet of samples was washed 13

ACCEPTED MANUSCRIPT twice with PBS and Fixed by adding 4.5 mL of cold ethanol (70%). After 24 h, the incubated cells were centrifuged (300×g for 10 min at4 °C), and was washed with PBS. After that, the suspended cells were stained with the mixture of ribonuclease A (500 µL, 100 µg.mL-1) and propidium iodide (500 µL, 100 µg.mL-1) and incubated at 37 °C for 1h. Cell population were

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analyzed to determine the cell cycle stage by flow cytometer.

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2.13.2. DAPI staining

The morphological evidence of apoptosis was monitored by DAPI nuclear staining method on

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MCF-7 cell line. Cells were cultured at density of 4×105 cell/well onto glass coverslips in six-

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well plates and incubated overnight. After that, the cells were treated with the mixture of free drugs DOX@MP and DOX@MP-Au/NGs (drugs concentration = 1 µg.mL-1). After 24h, the

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cells were fixed with fresh 4% paraformaldehyde for 30 min and the fixed cells were washed

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with PBS and incubated with DAPI stain solution (1 mg.ml-1) (Sigma, USA) for 30 min. The coverslips were probe using an inverted fluorescence microscopy (Bh2-RFCA, Olympus, Japan)

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and the stained cells were captured to observe the typical photographs.

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3. Results and discussion

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3.1. Development of ternary-responsive hybrid Au/NGs encapsulate two anti-cancer drugs Depending on the exterior triggers types, when the NGs are exposed to redox medium or acidic and hyperthermia conditions, the NGs would destruct to linear polymer chains or change the morphology of NGs to agglomerated forms, which leads to a quick drug release from the NGs structure. Thus, to synthesize the NGs with properties which mentioned above, we used: a) NIPAAM as thermo-responsive monomer [33], b) MAc as pH-responsive monomer and biocompatible crosslinker agent due to the existence of double bond in its structure [34], c) BAC 14

ACCEPTED MANUSCRIPT containing disulfide bond, used as redox-responsive monomer and a cross-linker to afford NGs GSH responsiveness [35,36] and d) encapsulated Au NPs to provide the best platform for load of drugs containing thiol groups such as MP in the core of NGs [37]. Additionally, PEGylation of NGs provides “stealth” characteristics for NGs otherwise is considered as foreign materials by

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human body [38]. The preparation procedure of hybrid Au/NGs including five steps is illustrated

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in Scheme 2. The first step was the synthesis of mPEG-Br macroinitiator. In the second step, the

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block copolymer of [PEG-b-P(NIPAAm-co-HEMA)] was synthesized. Third, the rings opening of MAn monomers was performed by pendant hydroxyl groups of HEMA [PEG-b-P(NIPAAm-

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coHEMA-g-MAc)]. Forth, the NGs were prepared by the polymerization of BAC as both of

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crosslinker agent and redox-responsive monomer and DMAEMA as ligand molecule to keep Au NPs inside NGs. After that, the in situ reduction of gold ions was carried out with NaBH4 in the

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presence of NGs in the fifth step. In conclusion, the main objective of this study is to design the

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core-crosslinked NGs that could respond to three stimuli triggers for drug modified release in tumor tissue. Finally, we assessed the ability of hybrid Au/NG to load of DOX and MP for

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potentialutility in the effective cancer therapy.

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Scheme 2. The steps in the synthesis of smart nanogels containing gold nanoparticles (Au NPs).

To study the chemical structure of copolymers, the FTIR and 1HNMR spectra were analyzed, as shown in Fig.1. For this purpose, the chemical composition of the synthesized copolymers was 16

ACCEPTED MANUSCRIPT studied by FTIR analysis, by which the spectrum of the PEG-b-P(NIPAAm-co-HEMA) copolymer showed two strong absorption peaks at about 1732 cm-1 and 1680 cm-1, which could be attributed to the carbonyl groups of HEMA and NIPAAm, respectively [38]. The stretching vibration of methylene groups in the PEG segment is represented by the strong absorption bonds

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at 2700–2950 cm-1. The ring opening reaction and grafting of MAc groups in the backbone of

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copolymer was confirmed by the presence of a new absorption band at 1710 cm-1, which is

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attributed to the carbonyl groups (carboxylic acid) of MAc (Fig. 1a) [39]. 1HNMR spectra exhibited sharp signals related to PNIPAAm, PHEMA, and PEG units (Fig. 1b). (Fig. 1b). The HNMR spectrum of the PEG-b-P(NIPAAm-co-HEMA) (in CDCl3) showed the peaks at: 3.65

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(4H, –OCH2CH2– of mPEG), 1.20 (6H, –C(CH3)2Br of BIBB), 3.30 (3H, CH3O– of mPEG), 4.1 (2H, –CH2–CH2–OH of PHEMA), 3.90 (2H, –COO–CH2– of PHEMA),. 1.60-2.10 (2H, –CH2–

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C(CH3)(COO)– of PHEMA and 2H, –CH2–CH2–CON– of PNIPAAm), 0.95-1.2 (3H, –

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CH2C(CH3)(COO)– of PHEMA and 6H, –NCH3– of PNIPAAm) and 1.42 ppm (2H, –CH2– CH2–CON– of PNIPAAm) [40]. The successful incorporation of MAc onto the backbone of the

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copolymer is characterized by signals at 6.28 and 6.86 ppm, which are attributed to the methane

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protons of the MA groups [39]. To obtain the average molecular weight (Mn) of the di-block copolymer, the integrals of signals at δ 3.8 (protons of PNIPAAm) and δ 1.42 (methylene

[41].

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protons of PHEMA) to δ 3.6 (methylene protons of PEG) were calculated to be 10.3 kg.mol-1

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Figure 1. FT-IR spectra of PEG-b-P(PNIPAAm-co-HEMA), PEG-b-P(PNIPAAm-co-HEMA-g-MAc)

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and PEG-[P(NIPAAM-co-HEMA-g-MAc)-BAC-co-DMAEMA] (nanogel) (a), 1H NMR spectra of PEG-

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b-P(PNIPAAm-co-HEMA) and PEG-b-P(PNIPAAm-co-HEMA-g-MAc) (b).

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One of the advantages of using NPs for drug delivery is because of their small size, which allows

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them to extravasate through the endothelium in inflammatory sites, epithelium (e.g., intestinal tract and liver), and tumors, penetrate microcapillaries, and lead to efficient uptake by a variety

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of cell types and selective drug accumulation at target sites [42]. Therefore, particle size and size distribution are the most important characteristics of NPs owing to the determination of in-vivo distribution, biological fate, toxicity, and targeting ability of these delivery systems [43]. Thus, PCS analysis was performed, which showed an average diameter of approximately 136 nm. This diameter is an ideal size of NPs for drug delivery to the cancerous tissues, because the suitable size range for EPR effect is 50-150 ~nm [44]. In addition to the size, size distribution is also a critical factor influencing the reproducibility in the accurate drug dose delivery to the target site 18

ACCEPTED MANUSCRIPT [45]. It has been reported that PDI < 0.2 represent the uniform size distribution [45] In this regard, the NGs developed in this work showed a PDI of 0.093 that is a quite ideal value (Fig. 2a). After entrapment of Au NPs, the size of hybrid Au/NGs (163 nm, PDI 0.102) did not change significantly and only became slightly larger than that of blank NGs (Fig. 2b). It was expected

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that the size of Au/NGs was dependent on the change of temperature due to the presence of

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thermoresponsive polymeric segment of NGs [46]. As shown in Fig 2b, at 25℃, the

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hydrodynamic diameter value of Au/NGs was 163 nm, whereas by raising the temperature to

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37℃ and 41℃, the PNIPAAm segment of the NGs collapsed and led to the shrinkage of NGs network. Therefore, as shown in Fig. 2b, the hydrodynamic diameter values decreased to 148 nm

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(37℃) and 129 nm (41℃). The zeta potential of NPs is commonly measured to characterize the surface charge property of NPs. This potential is known to be influenced by the composition of

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the particle and the medium in which it is dispersed [47]. Fig. 2c presents the zeta potential of

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Au/NGs in phosphate buffered saline (PBS). Despite the presence of positive and negative charges on the NPs surface because of the synthesized polymeric structure, the zeta potential

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value of NGs was almost 0 mV, which might confirm the successful PEGylation of NGs [48].

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Although PEGylation completely shield the surface charges of NGs, the steric effect of PEG chains caused colloidal stability of NPs because of preventing the particle aggregation and

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increased the circulation time of NGs in bloodstream. In addition, the TEM image of blank NGs displayed uniformly spherical shape of NGs with a diameter of 60-70 nm and illustrated the well dispersed NGs, in agreement with the PCS results (Fig. 2d). TEM image of Au/NGs after the reduction of gold ions in the presence of NGs is shown in Fig. 2e, where there is no significant change in the NGs size. Moreover, the hydrodynamic diameters of the blank and hybrid NGs

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the remove of physically or chemically adsorbed water molecules. The second weight loss

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between 120-200 °C might be related to the loss of MA side groups and PEG chains [34]. The major weight loss can be ascribed to the decomposition of PHEMA, PDMAEMA and BAC at

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about 220-380 °C, 340-400 °C and 200-400 °C, respectively [49–51]. The fourth weight loss between 400-450 °C was due to the loss of PNIPAAm chins [52]. To verify the successful

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entrapment of Au NPs in the NGs, the UV-vis spectroscopy spectra of blank NGs, Au NPs,

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MP@DOX-Au/NGs and hybrid Au/NGs were recorded (Fig. 2g). The results showed that Au

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NPs with the surface plasmon resonance (SPR) at 512 nm had a red shift in the SPR peak at 528 nm, suggesting the formation of Au NPs in the cavities of NGs, which also led to a decrease in

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the absorbance intensity of Au NPs [53,54]. Au NPs tend to thiol group in more degree than disulfide cross-lined bonds as shown in Fig. 2g, that the surface plasmon absorbance (SPA) bond

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of hybrid Au/NGs was appeared at approximately 528 nm. After the coupling of MP drug, the

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UV-Vis spectrum displayed a slight red shift (533) which might be associated the grafting of MP on the surface of Au NPs (Fig. 2g). After saturation of Au from MP, the rest may interact with disulfide cross-lined bonds. Even if the MP is bound by the thiol-exchange reaction to the disulfide bonds of the synthetic nanogels, supports the relatively high loading amount of MP. Moreover, since the number of these cross-linkers is high, replacing the MP in some of these connections will not be a serious problem in our hypothesis. Blank NGs revealed no sharp UV absorption above 400 nm. The colloidal stability of hybrid Au/NGs was confirmed by UV 20

ACCEPTED MANUSCRIPT spectroscopy results during the storage time (Fig. 2h). As reported in the literature [55], the maximum time for blood distribution of NPs is around 24 h but, for more certitude, the storage time was extended until 14 days for this test. The colloidal stability of Au/NGs did not change visually at all and the small changes in UV absorption of samples might be associated with the

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solvation of ionic functional groups of NGs, which caused a minor change in the conformation of

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PEG chains during 24 h. The observed ideal colloidal stability of Au/NGs might be attributed to

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the steric stabilization effect of the hydrophilic PEG tethered from the surface of the NPs. The thermal and pH sensitivity of hybrid Au/NGs were assessed through the alteration of the optical

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absorbance as a function of temperature and pH (Fig. 2i). With high rates of aerobic glycolysis

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and fast proliferation, temperature is higher (40–42 °C) in tumors than normal tissues [56–59]. Therefore, programmed temperature-responsive nanocarriers can be considered on the basis of

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the temperature difference between tumor tissues and normal tissues. Among the thermo-

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responsive polymers, Poly(N-isopropylacrylamide) (PNIPAAm) possesses a sharp phase

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transition and a low critical solution temperature (LCST), which is close to the body temperature (32℃). By increasing the temperature above the LCST of these polymers, a disruption of

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water-polymer hydrogen bonding and the intra/inter-chain hydrogen bonding occurs, which

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induce a change in the conformation of polymers to a collapse state [60]. Fig. 2i presents the LCST behavior of hybrid Au/NGs using spectrophotometer in the wavelength of 600 nm at concentration of 250 mg.L-1. The carboxylic groups of MAc made a pH-dependent LCST of Au/NGs. This behavior, which can be attributed to the protonation of the carboxylic groups of Mac, lead to a decrease in the hydrophilicity of the NGs. Therefore, by increasing the pH value from pH 6 to pH 7.4, the absorbance of the NGs solution was decreased and the LCST of PNIPAAm was increased from 41℃ to 47℃ [61].

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Figure 2. Size profiles of PEG-b-[P(NIPAAM-co-HEMA-g-MAc)-BAC-co-DMAEMA] nanogels (NGs)

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at 25 °C (136 nm, PdI: 0.093) (a), nanogels containing gold nanoparticles (hybrid Au/NGs) at 25 °C (163 nm, PdI: .0.102), 37 °C (148 nm, PdI: 0.139) and 41 °C (129 nm, PdI: 0.116) (b), zeta potential of hybrid Au/NGs at room temperature (c), TEM of NGs (d) and hybrid Au/NGs (e), TGA curve of hybrid Au/NGs (f), UV-vis spectra of NGs, pure Au NPs, hybrid Au/NGs and MP@DOX-Au/NGs in phosphate buffered saline (PBS) (g), investigation of long term stability of hybrid Au/NGs in PBS by the time dependence of absorbance (h), and temperature and pH responsive behavior of NGs as function of absorbance changes (i).

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3.2. Drug loading and release studies The capability for drug adsorption by NGs was evaluated to test the loading and controlled release behaviors of DOX and MP as cationic and thiol-containing anticancer drugs,

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respectively. DOX contains a positive charge in physiological pH condition (pH 7.4) due to the protonation of amino group of its structure (pKa = 8.3) [62]. In addition, the carboxylic acid

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groups of grafted PMAc are able to ionize at pH 7.4 due to the pKa value of PMAc; which is 6.2.

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Therefore, because of the electrostatic interaction between the protonated amino group of DOX and the carboxylate anion of the MAc segment, the EE and LC value of DOX were found to be

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quite high; i.e., 36.9% ± 4.1% and 11.53% ± 0.3%, respectively. It seems that the polymeric

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matrix of NGs stabilizes the ion-paired drug loading by reducing the thermodynamic activity of water. Because of the high affinity of sulfur groups for binding to the surface of the Au NPs in

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the form of direct attachment [63], the exciting high values EE (99.14 ± 5.32%) and LC (49.57 ±

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2.65%) were obtained for MP drug. The developed nanocarrier was designed to be responsive to the alterations of the oxidative and pH of environment. Thermo-, pH-, and redox-dependent

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release behavior of DOX@MP-Au/NGs was shown in Fig. 3. The cumulative release of drugs

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was shown in different simulated conditions. By comparing the profiles, it was seen that the release of DOX in simulated tumor tissue (Fig. 3a) and normal tissue (Fig. 3d) conditions was stimuli-responsive. Fig. 3a and 3d showed that 79% and 88% of DOX and MP was released for simulated cancerous environment (pH 6, 41℃, and 10 mM GSH), respectively and 25% and 12% of DOX and MP were released, respectively for normal tissue environment (pH 7.4, 37℃, and 2µm GSH). By comparing Fig. 3c with 3d, it was concluded that the contribution of temperature was almost 17% for both of drugs while by comparing Fig. 3b with 3d, it was indicated that the 23

ACCEPTED MANUSCRIPT contribution of pH was around 35% in DOX release. Only a negligible change (6 %) in MP release was occurred by pH change probably because of pH-dependent nature of S-Au bond [64,65]. The reason of participating pH as a main role in DOX release can be explained by the fact that the pKa value of MAc groups (6.2) was larger than the environment pH. The

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dissociation of the ionic bonds between the PMAc segment and DOX occurred due to the

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protonation of carboxylic anions in acidic pH. The temperature role was due to the collapse of

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the PNIPAAm segment and the consequent drug leakage from the polymeric lattice. The formed Au-S bond is stable in physiological conditions. Besides, the thiol-containing drugs bound on Au

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NPs surface can be exchanged by other thiols such as GSH [37]. GSH is often abundant in the

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tumor tissue and is used for releasing thiol-containing payloads bound on Au NPs [66,67]. Therefore, to study the redox-responsiveness of the prepared carrier, the release of MP was

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investigated at the elevated cancerous concentration (10 mM) and the physiological plasma

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concentration (2 µM) of GSH. By comparing Fig. 3d with 3f, it was concluded that the contribution of redox responsiveness on the release of MP and DOX was 61% and 24%,

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respectively. Finally, it can be concluded that this developed carrier leads to the fast drug release

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in the tumor environment and drug release control in the physiological environment.

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Figure 3. Cumulative in vitro release profiles of doxorubicin (DOX) and 6-mercaptopurine (MP) under different conditions from nanogels containing gold nanoparticles (DOX@MP-Au/NGs) (data are

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presented as mean ± standard deviation, n = 3).

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3.3.1. MTT assay

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3.3. In vitro biocompatibility study of developed hybrid Au/NGs

Via the MTT cell assay test, the biocompatibility of hybrid Au/NGs was assessed as a drug delivery system. As shown in Fig. 4a, the blank hybrid Au/NGs was first studied and no evident toxicity on the viability of MCF-7 cell line was revealed even in the highest amount of NGs for 48 and 72 h. This result demonstrates the good biocompatibility of the developed nanocarrier. 3.3.2. Hemolysis assay

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ACCEPTED MANUSCRIPT The hemolysis degree of developed NGs was investigated to confirm the safety of the developed PEGylated NGs,s biocompatibility during the long-term blood distribution (Fig. 4b). As reported in the literature, the hemolysis rate (HR) up to 5% is acceptable for biomaterials [68]. Therefore, the HRs of different concentrations of NGs was calculated, where a slight increase occurred in

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the hemolysis rate of the blood by increasing the NGs concentration. The distilled water was

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used as positive control and the apparent hemolysis was detected after contacting with RBCs.

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Generally, the non-hemolytic reactions were observed for the samples containing 500 (1.05%)

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and 1000 µg.mL-1 (2.8%) of NGs and observed that HRs were lower than 5% up to 1000 µg.mL. While, the sample containing 2000 µg.mL-1 revealed a hemolytic reaction with 6.8 % of HR.

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With our information, the maximum administration dose of used anti-cancer drugs is 50 mg which cause the blood concentration of this drugs calculate to be 10 µg.mL-1 in an IV infusion

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(the estimated blood volume = 5 L) [69]. Therefore, the concentration of needed carrier for

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loading these two drugs would be 500 µg.mL-1 for DOX and 100 µg.mL-1 for MP. In this regard, to ensure the blood biocompatibility of NGs, the least concentration value of NGs examined in

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this test was 500 µg.mL-1. The stability of MP@DOX-Au/NGs was investigated in PBS, water

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and cell culture due to the effect of salt concentration in stability which proved by monitoring the

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absorption spectra (Fig. 4c) [70]. As shown in Fig.4c, the incubation of MP@DOX-Au/NGs in PBS or culture medium did not cause significant change in absorption pattern of NGs in comparison to the water demonstrated the suitability of developed system for biological application. This colloidal stability of our developed system in ionic condition approves the main role of PEG chains. The steric stabilization effect of the hydrophilic PEG chains caused to protect the NPs from aggregation in physiological condition [71]. The size of nanocarriers was determined by PCS after 6 months and the results was almost the same as initial assessment.

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Figure 4. The biocompatibility of nanogels containing gold nanoparticles (hybrid Au/NGs) against human breast epithelial adenocarcinoma (MCF-7) cell line for 24 h and 48 h (a), Hemocompatibility test

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of hybrid Au/NGs treated with different concentrations of 500 µg.mL-1, 1000 µg.mL-1, and 2000 of Au/NGs, saline as negative control and deionized water as positive control (b). Error bars represent the

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standard deviation of three measurements. Stability of MP@DOX-Au/NGs in different mediums (water, PBS and culture medium) (c).

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3.4. Cellular internalization

3.4.1. Cell internalization assessment by fluorescence microscopy

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Since the DOX is a fluorescent drug, the cell penetration ability of DOX@MP-Au/NGs was

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visually investigated and compared with DOX@MP by a fluorescence microscopic (Fig. 5a). As shown in Fig. 5a, the cellular uptake of DOX@MP-Au/NGs was significantly higher than those

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of DOX@MP in MCF-7 cells, suggesting that drugs were expertly transported into the tumor cells using our developed nanocarriers. This result shows that the limited cellular uptake of DOX was improved by active endocytosis of NGs and this approach may also overcome multiple drug resistance (MDR) caused by drug efflux from the cancer cells [72,73]. Moreover, with the passage of time up to 3 h, the intensity of red fluorescence was enhanced and, consequently, the intracellular drug concentrations and the cell cytotoxicity increased. This evidence is compared

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ACCEPTED MANUSCRIPT with MTT results described in the next section representing the successful cell internalization of NGs. 3.4.2. Cell internalization assessment by flow cytometry

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The in vitro cellular uptake of Au/NGs was also investigated by flow cytometry analysis to

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confirm the fluorescence microscopy results. As shown in Fig. 5b, the MCF-7 cells were

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incubated with DOX@MP-Au/NGs for 1 h and 3 h and the cells without any treatment were used as a negative control. The profiles showed an almost similar uptake behavior compared to

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the obtained results of fluorescence microscopy images. Moreover, the cellular uptake ability

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was quantified for free form of drugs and drug-loaded nanocarriers. First, DOX@MP-Au/NGs exhibited a higher cellular uptake extent (67.89%) than those of DOX@MP (59.97%) during 1 h

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and (81.23%) and (64.69%) during 3h, respectively, which is due to the role of time in the

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cellular internalization. Moreover, the higher cellular uptake of DOX@MP-Au/NGs than those of DOX@MP proposed that this NGs facilitated circumvent multiple drug resistance (MDR)

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efflux, which is known as “stealth” endocytosis.

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Figure 5. Fluorescence microscopy images of treated human breast epithelial adenocarcinoma (MCF-7) cells with the mixture of doxorubicin and 6-mercaptopurine (DOX@MP) and drugs-loaded nanogels containing gold nanoparticles (DOX@MP-Au/NGs) after 1h and 3h (a) and cell uptake study of DOX@MP and DOX@MP-Au/NGs after incubation with human breast epithelial adenocarcinoma (MCF-7) cell line for 1h and 3h (b).

3.5. In vitro cell cytotoxicity 29

ACCEPTED MANUSCRIPT 3.5.1. MTT assay Multidrug resistance can provide when cancer cells develop mechanisms to resist classes of chemotherapeutic agents. Drug resistance mediated by P-glycoproteins is a major barrier in microtubule-targeting cancer chemotherapy [74].Thus, to dominate the P-glycoprotein pumps

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operation, drug-loaded nanocarriers are employed to attain the better therapeutic efficacy by

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escaping of P-glycoprotein drug efflux transporters [75]. This information well confirmed the cell internalization results reported in the previous section informing successful cell

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internalization of NPs. as a result, by increasing the intracellular drug concentrations, the cell cytotoxicity would be increased by time. As shown in Fig. 6, MTT cell assay was studied to

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investigate the therapeutic efficacy of developed nanocarriers by comparing the cytotoxicity

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profiles of the single free form of drugs, the mixture of two drugs, and drug-loaded nanocarriers. The results showed that the mixture of DOX@MP induced a higher cell cytotoxicity than single

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form of drugs for 24 and 48 h, while DOX@MPAu/NGs exhibited the same or even in some

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cases a better cytotoxicity as the mixture of two drugs. Therefore, these results can be valuable in lowering the administration dose of anticancer drugs and. As a result, the loaded drugs act in a

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similar level or better cytotoxicity as the mixture/single form of drugs by avoiding the side

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effects of DOX and MP. Moreover, to find the synergistic effect of two drugs, the combination effect of drugs was measured by calculating the combination index (CI) and found that the CI values were below 1 for both 24 h and 48 h times, suggesting the synergistic effect of DOX and MP [20].

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Figure 6. The cell viability study of human breast epithelial adenocarcinoma (MCF-7) cell after

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incubation of doxorubicin (DOX), 6-mercaptopurine (MP), the mixture of drugs (DOX@MP) and drugsloaded nanogels containing gold nanoparticles (DOX@MP-Au/NGs) for 24h (a) and 48h (b).

3.5.2. DAPI Staining

Among the typical appearances of apoptosis, chromatin condensation is one of the marker to identify the apoptosis pathway [76]. Therefore, to qualify the cell death mediated by apoptosis or necrosis, the DAPI staining method was used to assess the nuclear morphology of cells. As shown in Fig 7a, condensation of chromatin and nuclei fragmentation of cells were observed 31

ACCEPTED MANUSCRIPT after treatment with the free form of drugs (DOX@MP), revealing the apoptosis pathway. By comparing the free form of drugs (DOX@MP) and DOX@MP-Au/NGs, the DNA damage showed a higher level for the cells treated with DOX@MP-Au/NGs. In this regard, the cell shrinkage, DNA breakdown, and accumulation of the nuclear chromatin occurred with an

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increase in apoptotic cells nuclei. This result confirms the effective role of NGs in therapeutic

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performance of cancer. 3.5.3. Cell cycle arrest analysis

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The cell cycle arrest analysis was used to assess the effect of nanocarriers on the cell cycle of

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human breast cancer cells via the flow cytometry (Fig. 7b). The cell growth and preparation of chromosomes for replication occur during the G1 phase as a main stage in the cell cycle. When

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the cell leaves the cycle, the resting phase (Sub-G0/G1) takes place corresponding to the cell

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apoptosis pathway and then the percentage of apoptosis can be analyzed by the accumulation of cells in Sub-G0/G1 phase. In this method, the cell populations of dying and dead cells are

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specified by propidium iodide staining assessed by flow cytometry. As shown in Fig. 7b, the

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percentage of apoptotic cells for DOX@MP and Au/NGs are 11.68% and 6.5%, respectively. In comparison, the accumulation of cells in sub-G0/G1 region was enhanced after treatment with

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DOX@MP-Au/NGs up to 19.59%, indicating a significant increase in the percentage of cell population in terms of apoptotic cells. Therefore, we found that the inhibition of proliferation and growth of MCF-7 cells was influenced by DOX@MP-Au/NGs (10 µg.mL-1), leading to a more apoptotic effect than the free mixture of DOX@MP.

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Figure 7. DAPI staining of untreated human breast epithelial adenocarcinoma (MCF-7), treated with the mixture of drugs (DOX@MP) and drugs-loaded hybrid Au/NGs (DOX@MP-Au/NGs) (a) and effects of the mixture of drugs (DOX@MP), hybrid Au/NGs (without drugs) and drugsloaded hybrid Au/NGs (DOX@MP-Au/NGs) on cell cycle distribution of MCF-7 cells (human breast epithelial adenocarcinoma cell line) (b).

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ACCEPTED MANUSCRIPT 4. Conclusion In summary, we designed a new type of ternary-responsive core-crosslinked hybrid Au/NGs to simultaneously load two anticancer agents. The ability of this NGs was investigated for a hybrid cancer therapy. First, the temperature/pH/redox ternary-responsive NGs were successfully

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synthesized by copolymerization of HEMA and NIPAAm using PEG macroinitiator via ATRP

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method. Second, MAc groups were grafted on the backbone of copolymer by ring-opening reaction via pendant hydroxyl groups of HEMA to use as a biocompatible crosslinker agent. In

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the third step, the double bonds of MAc groups were used as crosslinker agents for the polymerization of BAC (as redox-responsive monomer) and DMAEMA (as ligand molecule to

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keep Au NPs inside NGs) to prepare the novel multi-responsive NGs. In the fourth step, the

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encapsulation of Au NPs was performed by in-situ reduction of HAuCl4 salts in the presence of NGs as a suitable platform for effective load of thiol-containing drugs such as MP (11%) via the

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Au-S bond. Moreover, hybrid Au/NGs could effectively encapsulate DOX (23%) due to the

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electrostatic force between DOX and PMAc segment. The synthesized hybrid Au/NGs had uniform spherical structures with the size of about 150 nm and narrow size distribution (PDI <

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0.14). Furthermore, the neutral charge of PEGylated NGs led to a minimized non-specific uptake

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by the cells. The developed DOX and MP loaded NGs exhibited a smart release behavior in response to temperature, pH, and thiolreducing agents such as GSH in simulated cancerous and normal tissue medium. In addition, hybrid Au/NGs enhanced the accumulation and penetration of DOX@MP into tumor cells assessed by various qualitatively and quantitatively cell uptake studies. These results suggested that the nanocarrier developed with temperature/pH/redox multiple sensitivity provides new insights for the rational design of optimal platforms for the intracellular co-delivery of therapeutic agents. 34

ACCEPTED MANUSCRIPT Acknowledgment The financial support from the Drug Applied Research Center, Tabriz University of Medical Sciences were gratefully acknowledged (Grant#95/94).

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights  A novel multi stimuli-responsive PEGylated hybrid gold/nanogels was prepared. hermo-, pH- and redox-responsive drug release from nanocarriers was observed. High amount of two anticancer (Doxorubicin and 6-mercaptopurine) drugs was loaded on the

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smart nanoparticles.

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High cytotoxicity of drug-loaded nanocarrier was observed against MCF-7 cancer cell line.

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