Novel thermoresponsive star-liked nanomicelles for targeting of anticancer agent

Accepted Manuscript Novel Thermoresponsive Star-Liked Nanomicelles for Targeting of Anticancer Agent Aliyeh Ghamkhari, Raana Sarvari, Marjan Ghorbani, Hamed Hamishehkar PII: DOI: Reference:

S0014-3057(18)30787-0 https://doi.org/10.1016/j.eurpolymj.2018.08.008 EPJ 8521

To appear in:

European Polymer Journal

Received Date: Revised Date: Accepted Date:

26 April 2018 2 August 2018 6 August 2018

Please cite this article as: Ghamkhari, A., Sarvari, R., Ghorbani, M., Hamishehkar, H., Novel Thermoresponsive Star-Liked Nanomicelles for Targeting of Anticancer Agent, European Polymer Journal (2018), doi: https://doi.org/ 10.1016/j.eurpolymj.2018.08.008

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Novel Thermoresponsive Star-Liked Nanomicelles for Targeting of Anticancer Agent

Aliyeh Ghamkhari1,2†, Raana Sarvari1†, Marjan Ghorbani3, Hamed Hamishehkar4*

1

Department of Chemistry, Payame Noor University, P.O. BOX: 19395-3697, Tehran, Iran.

2

Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. .

4

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

*Corresponding author: Hamed Hamishehkar: Email address: [email protected]; [email protected]

†

These authors made equal contributions to the work.

1

Abstract Micelles are frequently used as drug carriers in the area of nanomedicine owing to their high potential for cancer therapy. In this study, we report the synthesis of a novel thermoresponsive star-liked

amphiphilic

block

copolymer

based

on

poly(ɛ-caprolactone)-b-poly(N-

isopropylacrylamide) [HAPs-g-PCL-b-PNIPAM] by ring-opening (ROP) and reversible addition fragmentation chain transfer (RAFT) polymerization. The micellar properties and thermoresponsive behavior of HAPs-g-PCL-b-PNIPAM were investigated by Transmission electron microscopy (TEM), Field emission-scanning electron microscopy (FE-SEM), ultraviolet-visible (UV-Vis) spectroscopies, dynamic light scattering (DLS) and Differential scanning calorimetry (DSC) measurements. We developed a biodegradable star-liked polymeric micelle for the overcome limitations of docetaxel (DTX)-loading and to enhanced pharmacokinetics. The DTX-encapsulation efficiency was obtained to be 95.5%. Release behaviors of DTX from the nanomicelles demonstrated that the rate of DTX release could be efficiently controlled by temperature and pH value. We demonstrated the cytotoxicity of the drug in vitro against breast cancer cell line (MCF7) using the MTT assays, DAPI staining, and cellular uptake. In conclusion, we visualized that the synthesized DTX-nanomicelles can be used as an anticancer drug delivery system considering their useful biocompatibility and excellent physicochemical properties.

Keywords: Thermoresponsive; Star-liked block copolymer; Docetaxel; Cancer therapy.

2

1. Introduction Drug delivery systems (DDSs) based on nanoparticles are vastly considered because of their ability in raising synergistic actions, reducing drug side effects, and improving target selectivity [1, 2]. These features are suitable to arrive high cellular uptake and “passive targeting” functions to the tumor supposed enhanced permeability. Thus, nanocarrier systems are potentially selected to direct drug delivery to the tumor tissue [3-6]. The self-assembled nanomicelles of amphiphilic molecules have nanoparticle size ranged from 10 to 100 nm [7-8]. Moreover, nanomicelles are used to enhance the solubility of low and insoluble drugs, protect and stabilize responsive drugs, extend the circulation time in the blood, and diminish the nonspecific uptake by the reticuloendothelial system (RES) [9, 10]. Docetaxel (DTX) is one of the most potent and hydrophobic anticancer drugs that its weak water solubility has forced the formulators to offer injectable dosage forms enriched with surfactants that have further side effects and finally restrict the clinical application of DTX [11-14]. Nanomicelles are composed of linear copolymers that have low performance under in vivo conditions because of an easy removal from the body by phagocytosis of reticuloendothelial system [15]. To overcome this problem, copolymers with hyperbranched or dendritic structures have been extended owing to their unique physicochemical properties including low viscosity, good solubility, and multi-functionality [16-18]. Hyperbranched compositions are considered to resist the phagocytosis by the development of unimolecular nanomicelles form [19, 20]. Among various hyperbranched structures, the hyperbranched aliphatic polyesters (HAPs) based on 2,2-bis(methylol) propionic acid (bisMPA) are very famous because of their extremely branched structures, unique physicochemical properties including a low flexibility, having a lower degree of crystallinity, and a great solubility in organic solvents [21-25].

3

From the aliphatic polyesters family, poly(ɛ-caprolactone) (PCL) is widely used as the hydrophobic segment with characteristics such as good biocompatibility, biodegradability, low immunogenicity, and controlled drug delivery [26, 27]. Stimuli-responsive nanomicelles can respond to a superficial stimulus including pH, temperature, and ionic strength [28- 30]. Among the stimuli-responsive block copolymers, poly(N-isopropylacrylamide) (PNIPAM) has been applied as a thermoresponsive polymer [31-33]. PNIPAM demonstrates a lower critical solution temperature (LCST) around human body temperature (32-34℃) in aqueous solutions, making it qualified for drug delivery applications [34, 35]. In addition, temperature- and pH-responsive star-like copolymers have potential applications in the controlled drug delivery to linear copolymers [36, 37]. The high molecular weight with shorter chains leads to a quicker degradation rate of the starlike copolymers to the linear copolymers and, associated with PCL segments, forms star-like amphiphilic block copolymers [38]. The aim of the present work is to design, synthesize, and characterize a novel thermoresponsive star-liked amphiphilic block copolymer (HAPs-g-PCL-b-PNIPAM) via ROP and RAFT polymerization. The micellar properties of copolymer were investigated by TEM, FESEM, and DLS, followed by studying the loading capacity and the drug release ability of HAPs-g-PCL-b-PNIPAM nanomicelles. Finally, the cytotoxicity studies of nanocarrier were performed using the MTT assay, DAPI staining, and flowcytometry analysis. 2. Experimental 2.1. Materials The RAFT agent (4-cyano-4-[(phenylcarbothioyl) sulfanyl] pentanoic acid) was synthesized in our laboratory [41]. Tris (methylol) propan (TMP), p-toluenesulfonic acid (p-TSA) and 2,2-bis(methylol) propionic acid (bis-MPA), were purchased from Fluka (USA). (ε4

caprolactone) (CL), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-nyltetrazolium bromide), 4,6-diamidino-2-phenylindole (DAPI), Fluorescein and other biological reagents were purchased from Sigma Aldrich (USA). N-Isopropylacrylamide (NIPAM, 97%, SigmaAldrich, USA) was purified by recrystallization in n-hexane/toluene mixture before use. The initiator of 2,2-azobisisobutyronitrile (AIBN; Fluka, Switzerland) was recrystallized in absolute ethanol at 50 °C. Docetaxel was purchased from TCI Company (Tokyo, Japan). All other reagents were purchased from Merck and purified according to standard methods. 2.2. Synthesis of hyperbranched aliphatic polyesters (HAPs) A mixture of Tris (methylol) propan (TMP) (0.28 g, 2.2 mmol), 2,2-bis(methylol) propionic acid (bis-MPA) (8.0 g, 46.4 mmol) and a catalytic amount of p-TSA (0.05 g, 0.28 mmol) were charged in a three-necked flask. The reaction mixture was degassed with argon and transferred in an oil bath at 140 °C under stirring to remove the water instituted during the reaction process. After 3 h, the glass-like product was obtained dissolved in acetone, and precipitated into cold hexane, then dried under vacuum (scheme 1a). 2.3. Synthesis of star-liked PCL (HAPs-g- PCL) The of hyperbranched aliphatic polyesters (HAPs) (0.3 g, 0.16 mmol) was dried at 110 °C. Then ɛ-Caprolactone (8 g, 70.35 mmol) and 0.4% Sn (Oct)2 in toluene solution (10 mL) was refluxed at same temperature for 24 h. The resulting combination was dissolved in CH 2Cl2, and precipitated into cold hexane, then dried in vacuum (scheme 1b). 2.5. Synthesis of star-liked RAFT agent (HAPs-g-PCL-RAFT). In to a two-necked flask the HAPs-g-PCL (1 g, 0.05 mmol) and RAFT agent (0.45 g, 1.6 mmol) were dissolved in 20 mL of dried dichloromethane (CH 2Cl2). Then, the solution containing DCC (0.5 g, 2.5 mmol), dimethylamino pyridine (DMAP) (0.35 g, 1.2 mmol) and 5

dichloromethane (30 mL) was added to the vigorously stirred reaction solution. The esterification reaction was made at 25 °C for 48 h. After performance of the reaction, the dicyclohexylurea (DCU) was removed by filtration. The resulting combination was dissolved in ethyl acetate and precipitated with diethyl ether, then dried in vacuum (scheme 1c). 2.6. Synthesis of star-liked hyperbranched aliphatic polyesters-g-poly (ɛ-caprolactone)– poly(N-isopropylacrylamide) [HAPs-g-PCL-b-PNIPAM] HAPs-g-PCL-RAFT (0.3 g, 0.15 mmol), N-isopropylacrylamide (0.6 g, 5.3 mmol) and DMF (6 mL) were dissolved in a two-necked flask. The reaction mixture was degassed with four freeze–pump–thaw cycles, and refluxed at 80 °C for about 48 hours. After performance of the reaction, the flask was transferred in ice bath, in order to quenching the polymerization. The reaction was precipitated in cold diethyl ether (150 mL). The product was filtrated and dried under vacuum at room temperature (scheme 1d).

6

7

Scheme 1. Synthesis of hyperbranched aliphatic polyesters (HAPs) (a), synthesis of star-liked HAPs-g-poly (ɛ-caprolactone) (HAPs-g-PCL) (b), synthesis of star-liked HAPs-g-PCLRAFT agent (HAPs-g-PCL-RAFT) (c) and synthesis of star-liked HAPs-g-PCL-poly(Nisopropylacrylamide) [HAPs-g-PCL-b-PNIPAM] (d). 2.7. Characterization The size exclusion analyses were carried out using a Waters 1515 (USA) gel permeation chromatography (GPC) instrument equipped with Breeze 1515 isocratic pump and 7725 manual injector. Fourier transform infrared (FT-IR) spectra of the samples were obtained in a Shimadzu 8101M FT-IR (Shimadzu, Kyoto, Japan) at the wavenumber ranges of 4000 to 400 cm–1. Proton nuclear magnetic resonance (1HNMR) and carbon-13 nuclear magnetic resonance (13CNMR) spectra were obtained at 25 °C using an FT-NMR (400 mHz) Bruker spectrometer (Bruker, Ettlingen, Germany). The samples were prepared in the deuterated dimethyl sulfoxide solvent (DMSO-d6). Ultraviolet-Visible (UV-Vis) spectra were taken in a Shimadzu 1650 PC UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). The particle size of nanomicelles was measured by a laser-scattering technique (Zetasizer Nano ZS90; Malvern Instruments, Malvern, UK) at 25, 35 and 41°C. The field emission scanning electron microscope (FESEM) type 1430 VP (LEO Electron Microscopy Ltd, Cambridge, UK) was applied to determine the morphologies of nanomicelles. The powder sample was spread on a SEM stub and sputtered with gold. Transmission electron microscopy (TEM) images of nanomicelles were taken on a Philips CM10-TH microscope (Phillips, Eindhoven, Netherlands) with a 100kV accelerating voltage. The Differential scanning calorimetry (DSC) measurement was performed on a TA Q20 calorimeter system under flowing nitrogen gas to determine melting point (Tm) and LCST of star-liked polymer and nanomicelles at a heating rate of 10 and 5 °C/min, respectively.

8

2.8. Preparation of DTX-nanomicelles DTX- nanomicelles were prepared by using a membrane dialysis method. 100 mg of HAPsg-PCL-b-PNIPAM copolymer was dissolved in 4 mL DMSO to prepare polymer solution. Then, DTX (10 mg) was added to mixture of the flask and stirred for 24 h under dark conditions. The solution was dialyzed against DI water (200 mL) at 20 °C for 48 h under dark condition. Then, DTX-nanomicelles mixture was collected by centrifugation at 10000 rpm for 20 minutes. The mixture was collected and used for calculating the encapsulation efficiency (EE) and loading content (LC) of DTX by the following equations:

9

Scheme 2. Schematic structure of docetaxel (DTX)-loaded hyperbranched aliphatic polyesters-g-poly(ɛ-caprolactone)–poly(N-isopropylacrylamide) (HAPs-g-PCL-b-PNIPAM) micelles. 2.9. In vitro release study In the in vitro drug release experiment, DTX-loaded nanocarrier (50 mg) was dispersed in PBS with pH values (pH = 7.4 and 5.4), then each 1.5 mL of dispersion was placed in PBS solution containing 1% Tween 80, which was applied to enhance the solubility of the DTX and establish the sink condition. The release solution was stirred at 250 rpm individually at 37 and 41 °C, and then 1.5 mL of buffer solution was collected in different times to evaluate characterization with UV-Vis spectrophotometer at 256 nm, instead, a fresh buffer solution

10

was added back to maintain the same total solution volume. The percentage of the cumulative amount of released DTX was calculated from the standard calibration curve. 2.10. In vitro cytotoxicity study The human breast adenocarcinoma (MCF7) and healthy normal embryonic fibroblast (3T3) cell lines purchased from NCBI (National Cell Bank of Iran, Pasteur Institute) and cultivated in RPMI1640 having 100 IU penicillin per 100 mg streptomycin, enriched with 10% (v/v) of fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). The cells were cultured into flasks and kept in a humidified incubator with 5% CO 2 at 37 °C and the media refreshed twice weekly. The cytotoxic effects of DTX-nanomicelles and free DTX were compared using of the MTT method on MCF7 cells. Briefly, the MCF7 cells were seeded in 96 well plates and after incubation for 24 h, the cells were treated with different concentrations (2.5, 5, 10, 20, 40 µg mL–1) of nanomicelles, DTX-nanomicelles, and free DTX for 24, 48 and 72 h. After that, the culture medium was removed and the MTT solution (5 mg/ml) was added to each well followed by 150µL cultivation medium, then cells were incubated for 4 h. for 4 h. Finally, the remaining MTT solution aspirated, the formed formazan crystals were dissolved in DMSO and the optical density was measured at 570 nm using a micro-plate reader (Elx808, Biotek, USA), and the results were compared with respect to control cells. 2.11. DAPI staining study A type of fluorescent dyes is DAPI (4, 6-diamidino-2-phenylindole) which bound strongly to the chromatin and subsequently more fluorescent is shined upon bounding. The DAPI nuclear staining was investigated to probe the apoptosis and to monitor the effect of DTXnanomicelles on MCF7 cell nuclei. Cells were seeded on glass coverslips and after incubation for 24 h at 37 °C, the cells were treated with IC50 doses of free DTX and DTX-nanomicelles for 24 h. After 48 h incubation, the cells were washed four times with PBS and fixed with 11

fresh 4% paraformaldehyde for 40 min at room temperature. Then, the cells nuclei were stained with DAPI solution (Sigma, USA) for 40 min. Finally, the stained cells were observed using a fluorescence microscopy (Bh2-RFCA, Olympus, Japan). 2.12. Cellular uptake study Fluorescein was labelled to DTX-nanomicelles to study the cellular uptake in MCF7 cells. To prepare fluorescein labelled nanomicelles, a solution of 2 mg/ml of fluorescein in DMSO was prepared. Then, 200 µl of this solution was added to 10 mg/ml solution of nanomicelles in PBS and stirred overnight at room temperature. Afterwards, the fluorescein labelled nanomicelles was collected by centrifuge at 8000 rpm for 5 min and washed several times to remove the unloaded fluorescein. MCF7 cells were seeded in 6 well plates at the density of 5×105 per well and incubated for 24 hrs. Cells at the density of 70 % confluence were treated with fluorescein labelled DTX-nanomicelles. After 1 and 3 h, cells were washed with PBS and trypsinized to examine with FACS calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) to measure the fluorescent intensity related to fluorescein uptake inside the cells. 3. Results and discussion Despite the extended clinical use of DTX chemotherapeutics, they have some systemic side effects. The star-liked biodegradable nanomicelles have been used for overcoming the restrictions of drug loading, weak serum stability, and enhanced pharmacokinetics of DTX [42, 43]. Herein, thermoresponsive HAPs-g-PCL-b-PNIPAM nanomicelles were designed by ROP and RAFT technique for drug delivery purposes. Then, the ability of this nanocarrier was studied against cancer cell line (MCF7) using the MTT assay, DAPI staining, and cellular uptake. 3.1. Characterization

12

The FTIR spectrum of the hyperbranched aliphatic polyesters (HAPs) revealed the characteristic absorption bands of ester carbonyl group at 1722 cm-1, stretching vibrations of aliphatic C-H at 2955-2800 cm-1 region, C-H bending vibrations at 1375 and 1469 cm-1, and C-O stretching vibrations at 1042 and 1215 cm-1. The broad strong band at 3323 cm-1 resulted from the hydroxyl groups of hyperbranched polyester. The successful synthesis of the HAPsg-PCL was confirmed by the creation of characteristic absorption bands of the symmetric stretching vibrations of C-O-C at 1249 and 1175 cm−1, ester carbonyl group at 1726 cm-1, the stretching vibrations of aliphatic C-H at 2932 cm-1 region, and broad strong band centered at 3490 cm-1 as hydroxyl end groups. The successful synthesis of HAPs-g-PCL-RAFT was demonstrated by the appearance of a new band at 2254 cm−1 related to cyanide group of RAFT moiety and the disappearance of hydroxyl stretching vibration band at 3288 cm−1. The most bands in the FTIR spectrum of the HAPs-g-PCL-b-PNIPAM were the stretching vibrations of aliphatic C-H at 2975-2855 cm−1, the stretching vibrations of ester carbonyl groups at 1726 cm−1, amid carbonyl groups at 1655 cm−1, and secondary amid stretching vibration as a broad and strong band centered at about 3321 cm−1 (Fig. 1). The successful synthesis of hyperbranched aliphatic polyesters (HAPs), HAPs-g-PCL, and HAPs-g-PCL-bPNIPAM were further described by means of 1HNMR spectroscopy. The 1HNMR spectrum of the hyperbranched polyester (HAPs) showed the peaks with the following shifts: The unreacted linear and terminal hydroxyl groups (OHL and OHT) disappear at 4.75 and 4.45 ppm, respectively. The main chemical shifts at 3.95 and 3.35 ppm correspond to the CH2OR (OR: OH reacted) and CH2OH (overlapped with H2O), respectively (Fig. 2A). The chemical shifts at 0.90 to 1.45 ppm are related to the methylene and methyl groups in the sample. In the 1HNMR spectrum of HAPs-g-PCL, the most chemical shifts include the chemical shifts at 4.05 ppm (f) and 2.3 (c), which are indexed to the protons of (-CH2-O-) and (-CH2-CO-), respectively (Fig. 2B). The chemical shifts at 1.6 ppm (d) and 1.4 ppm (e), which are indexed

13

to the -CO-CH2-CH2-CH2-CH2-CH2-O- and -CO-CH2-CH2-CH2-CH2-CH2-O- protons, respectively. The characteristic chemical shifts of the HAPs-g-PCL-b-PNIPAM are clearly observed. The chemical shifts at 1.04 (i) 1.9 (g) and 3.9 (h) were ascribed to the methyl and methylene protons and methyne proton of N-CH-, respectively. Furthermore, the shift at 7.17.3 (k) ppm is related to the aromatic protons (Fig. 2C). In addition,

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CNMR spectrum of

HAPs-g-PCL-b-PNIPAM (Fig. 2D) showed the chemical shifts at 21.3 (CH3-C), 23.5-28.0 ppm (CH2 – CH2) and (CH-NH), 32.0-33.2 ppm (CH2-CO), 61.45 ppm (HAPs), 63.30 ppm (CH2-O), 75.6-76.3 ppm (CDCl3), 122.7 ppm (C=C aromatic) and 172.5 ppm (CO). The morphology of the HAPs-g-PCL-b-PNIPAM nanomicelles was investigated using TEM and FESEM analyses. The TEM image of HAPs-g-PCL-b-PNIPAM nanomicelles was captured by staining with a phosphotungstic acid agent in Fig. 3a. The morphology of nanomicelles showed spherical shapes with mean diameters of about 25 nm. The SEM image of HAPs-g-PCL-b-PNIPAM nanomicelles is presented in Fig. 3b. The HAPs-g-PCL-bPNIPAM nanomicelles had spherical shapes and an average diameter of 45±10 nm. PNIPAM is one of the mostly used temperature-responsive polymers, which exhibits a low critical solution temperature (LCST) at around 32℃. By increasing the temperature above LCST, it creates opportunities for biomedical applications [44]. UV-Vis spectroscopy was used to conduct LCST measurements. The sample was solvated at 30℃, while the transmittance of UV-Vis light was measured as a function of temperature. The nanomicelles showed LCSTs at 38-40℃ (Fig. 3c). DLS studies also confirmed the thermosensitivity of hyperbranched nanomicelles. The particle size of nanomicelles was analyzed by DLS at different temperatures. The average sizes of the HAPs-g-PCL-b-PNIPAM nanomicelles at temperatures of 25, 35, and 41℃ were measured to be 88, 107, and 62 nm, respectively (Fig. 3d). As a result, the particles size decreased to 62 nm when the temperature exceeded its

14

LCST temperature (41℃); leading to the collapse of PNIPAM chains and shrinkage of the nanomicelles network [45, 46]. TEM image is obtained in the absence of the solvent while the hydrodynamic diameter of the solvent is determined using DLS technique. Thus, the average sizes of the particles obtained from the TEM analysis were smaller than that DLS [41]. Thermal properties of the star-liked HAPs-g-PCL-b-PNIPAM copolymer and nanomicelles were also investigated by DSC analysis (Fig. 4 (a,b). The synthesized star-liked copolymer shows an obvious crystallization peak (=5.6 oC) and a melting peak (= 40.6 oC) at cooling and heating cycles, respectively (Fig 4a). As can be seen in fig. 4b, single endothermic DSC peak appeared in heating at 39.1 °C, ascribing to LCSTs of the nanomicelles. The GPC chromatograms of the HAPs-g-PCL and HAPs-g-PCL-b-PNIPAM samples are shown in Table 1. The PDI values of the HAPs-g-PCL and HAPs-g-PCL-b-PNIPAM synthesized via ROP and RAFT polymerization are PDI=1.52 and PDI=1.58, respectively. The molecular weights of HAPs-g-PCL-b-PNIPAM showed a relatively low polydispersity, suggesting a good control over the RAFT polymerization. The molecular weights obtained from GPC and HNMR are compared in Table 1.

15

Fig. 1. FTIR spectra of the hyperbranched aliphatic polyesters (HAPs), star-liked HAPs-gpoly (ɛ-caprolactone) (HAPs-g-PCL), star-liked HAPs-g-PCL-RAFT agent (HAPs-g-PCLRAFT), star-liked HAPs-g-PCL-poly(N-isopropylacrylamide) [HAPs-g-PCL-b-PNIPAM].

16

17

Fig. 2. 1HNMR of the hyperbranched aliphatic polyesters (HAPs) (A), star-liked HAPs-gpoly(ɛ-caprolactone)

(HAPs-g-PCL)

(B),

star-liked

isopropylacrylamide) [HAPs-g-PCL-b-PNIPAM] (C) and g-PCL-b-PNIPAM] (D).

18

13

HAPs-g-PCL-poly(N-

CNMR of the star-liked [HAPs-

Fig. 3. Transmission electron microscopy (TEM) micrograph of micelles obtained using phosphotungstic acid staining (a) and field emission scanning electron microscope (FESEM) image (b), LCST of nanomicelles (c), and Temperature dependent particles size of nanomicelles (d).

19

Fig. 4. Differential scanning calorimetry (DSC) thermograms of star-liked [HAPs-g-PCL-bPNIPAM] copolymer (a) star-liked [HAPs-g-PCL-b-PNIPAM] nanomicelles (b).

20

Table 1. Molecular weight analysis data of HAPs-g-PCL and HAPs-g-PCL-b-PNIPAM samples by GPC and 1HNMR. Mn a(GPC)

Mn b(1HNMR)

Mwa

PDIa

-

1808

-

-

HAPs-g-PCL

19765

20345

30091

1.52

HAPs-g-PCL-b-PNIPAM

47300

44905

74700

1.58

Sample HAPs

Mn; number average molecular weight, Mw; weight average molecular weight, PDI; Polydispersity; a. Determined by GPC; b. Determined from 1HNMR data. 3.2. Drug loading and in vitro release behavior study of DTX-nanomicelles To improve the biodistribution and pharmacokinetics of drugs, star-like nanocarriers were developed for drug delivery systems. The main justification for use of these systems is that they provide a controlled release of drug with the goal of reaching to the target tissues. To study the in vitro release behavior of nanomicelles, we used DTX as a representative microtubule-stabilizing chemotherapy drug [47, 48]. LC and EE of DTX were calculated to be 9.5 and 95.5%, respectively. The surface morphology of DTX-nanomicelles was represented by FE-SEM in Fig. 5a. The DTX-nanomicelles demonstrated morphology of spherical nanomicelles with an average diameter of 50±10 nm. The pH responsive of DTXnanomicelles was investigated by DLS. The effect of pH on the hydrodynamic diameter of DTX-nanomicelles was plotted in Fig. 5b. At pH=7.4 and T=37℃ diameter of the DTXnanomicelles a smaller hydrodynamic diameter (93.4 nm) was observed. At pH=5.4 and T=37℃ an increase in the diameters (109 nm) was observed. As can be seen from the release profile at 37℃ and 41℃, DTX release from nanomicelles was a pH-responsive process. According to Fig. 5c, the release in pH values (5.4 and 7.4) after 222 h was 58.16 and 41.38 %, respectively. The release property of DTX-micelles demonstrated a sharp sustained 21

release mode compared with the release process of the free DTX solution. Although 35% DTX was released from the nanomicelles in the primary 6 h, the fast release would not influence the drug safety. The standard of a burst release is defined as more than 40% of the drug being released in 0.5 h according to the Chinese Pharmacopoeia [12], while less than 20% of the drug was released from the nanomicelles during 1 h, which did not reach the burst release level. Less than 10% of DTX was released in physiological condition (pH 7.4 and temperature of 37 oC) (Fig. 5d) during 1 hour (first sampling time). In real condition, nanoparticles were distributed and accumulated in cancerous tissue by “enhanced permeability effect” phenomena after a few seconds or maximum few minutes of intravenous injection. Therefore, we believe that even much less than 10% of DTX will be released from nanoparticles after IV injection. Therefore, a 35% release during 6 h would not affect the drug safety. The thermoresponsive carrier under a structural transition as a response to increasing temperature leads to the certification of the drug and drug easier absorption by cells [49, 50] [44]. Thus, the release profiles at 41℃ in pH values of 5.4 and 7.4 showed that by increasing the temperature, the drug release values were accelerated to 63.64 and 46.67%, respectively. This phenomenon may occur due to the collapse of the thermo-responsive segment (PNIPAM) at above LCST [44, 51] of nanomicelles owing to the compact loading of DTX in the micellar core.

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Fig. 5. Field emission scanning electron microscope (FESEM) image of DTX-nanomicelles (a), Dynamic Light Scattering (DLS) diagram of the DTX-nanomicelles at pH=7.4 and 5.4 , T= 37 °C (b), In vitro release profiles of docetaxel (DTX) from DTX-nanomicelles at various temperatures (T=37 and 41 °C) (c, d). 3.3. In vitro cytotoxicity The in vitro cellular cytotoxicity of the DTX-nanomicelles against MCF7 cells and 3T3 were investigated using an MTT assay. The plain nanomicelles showed no cytotoxic effects even after incubation with 40 µM for 24, 48, and 72 h. As shown in Figure 6(a, b, c, d), the cell viability of micelles showed no significant toxicity on viability of MCF-7 (as cancer cell line) and 3T3 (as normal cell line) cells after 24 h, 48 and 72 h, confirming that the nanomicelles exhibited a good biocompatibility.

As can be seen from Fig. 6 (a, b, c), when the

concentration of the DTX-nanomicelles was 40 µM, the viability of cells treated with DTX23

nanomicelles significantly decreased. DTX-nanomicelles displayed a cytotoxicity about 10% higher than that of free DTX after 72 h at concentrations more than 10 µgmL -1. It can be further noticed that the IC50 values of the DTX formulated in the nanomicelles after 72 h treatment for DTX and DTX-nanomicelles were 5 and 3.75 µM, respectively. Moreover, the results revealed that by increasing the concentrations and times, the cell viability percentages were decreased due to the time and dose-dependent release of DTX. As shown in Fig. 6e, apoptosis properties of the DTX-nanomicelles were compared with free DTX against MCF7 cells using the DAPI staining assay. The morphological changes of DTX-nanomicelles were shown with condensation of chromatin in nuclear and fragmentation of nuclei; i.e., apoptosis. Accordingly, the frequency of chromatin fragmentation in DTX-nanomicelles treated cells was more than that of free DTX. Thus, it can be concluded that the DTX-nanomicelles will be a superior candidate for controlled drug delivery applications.

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Fig. 6. The cell viability study of docetaxel (DTX)-nanomicelles, nanomicelles and free DTX with different concentrations (2.5, 5, 10, 20, 40 µgmL -1) in time period of 24 (a), 48 (b) and 72 (c) hours against human breast epithelial adenocarcinoma (MCF7) cell line and the biocompatibility test against the embryonic fibroblast cells (3T3) as normal cell line (d). DAPI staining of untreated human breast epithelial adenocarcinoma (MCF7) cells (control), treated with DTX and DTX-nanomicelles (e) 3.4. Cell internalization Fluorescein-labeled nanomicelles was employed to exhibit the percentage of cellular uptake. The flowcytometry analysis of MCF7 cells treated with Fluorescein-labeled nanomicelles is shown in Fig. 7a. The cells without any treatment were considered as negative control. Fluorescein-labeled DTX-nanomicelles indicated the cellular uptake of DTX-nanomicelles in the cells after 1h and 3 h incubation. The results show that the nanomicelles can be effectively transported into tumor cells during 3 h, suggesting that DTX-nanomicelles can be 25

applied as a potential nanocarrier for cancer therapy. Herein, DTX can be applied as a microtubule-stabilizing chemotherapy drug loaded in nanomicelles. The fluorescence microscopy was assessed for further investigation in the capability of nanomicelles in the cell penetration. As shown in Fig. 7b, there is a little green fluorescence in the MCF7 cells after 0.5 h and 1 h of incubation and strong green fluorescence was detected after 3h of incubation by increasing the cellular uptake of DTX-nanomicelles. These images suggest that the nanomicelles can transport competently drug into tumor cells during 3 h, proving the obtained result of flowcytometry analysis.

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Fig. 7. Cellular uptake images of human breast epithelial adenocarcinoma (MCF7) cells after incubation with fluorescein labelled docetaxel (DTX)-nanomicelles after 1 and 3 h (a) and fluorescence microscopy images of treated MCF7 cells with the docetaxel (DTX)nanomicelles after 0.5, 1 and 3 h (b). 4. Conclusion We developed some thermoresponsive star-like hyperbranched aliphatic polyesters by following steps. First, star-liked HPAs-g-PCL was prepared by a macroinitiator via the ringopening polymerization of CL. Then, HAPs-g-PCL-RAFT was synthesized using the 27

esterification of HAPs-g-PCL with RAFT agent using DCC as the coupling agent. Finally, the NIPAM monomer was polymerized in the presence of HAPs-g-PCL-RAFT to prepare the HAPs-g-PCL-b-PNIPAM nanomicelles. The molecular weights of HAPs-g-PCL and HAPsg-PCL-b-PNIPAM were estimated to be 19765 and 47300 g/mol, respectively. The morphology of HAPs-g-PCL and HAPs-g-PCL-b-PNIPAM nanomicelles had spherical shapes with mean diameters of about 25 nm and 45±10 nm by TEM and FESEM, respectively. The LCST of HAPs-g-PCL-b-PNIPAM nanomicelles was measured at 38-40℃ and by increasing the temperature to 40℃, the size of HAPs-g-PCL-b-PNIPAM nanomicelles was decreased to 62 nm. DTX-loading capacity and DTX-encapsulation efficiency were calculated to be 95.5%. The obtained release profiles at 37℃ indicated that 58.16 and 41.38 % of DTX was released at pH values of 5.4 and 7.4, respectively. In comparison, by increasing the temperature to 41℃ in pH values of 5.4 and 7.4, the release rates were increased to 63.64% and 46.67%, respectively, which confirmed the thermos-responsitivity of the developed nanomicelles. Temperature and pH dependent release pattern was confirmed. The biocompatibility of the nanomicelles was approved through the MTT assay. Additionally, the cytotoxicity of DTX-nanomicelles was investigated and found that the nanomicelles had a significant anticancer performance compared with free DTX; verified by the MTT assays, DAPI staining, and intracellular uptake. Acknowledgment The financial support from the Drug Applied Research Center, Tabriz University of Medical Sciences were gratefully acknowledged. Reference 1. K. Kataoka, A. Harada and Y. Nagasaki, Block copolymer nanomicelles for drug delivery: design, characterization and biological significance. Advanced Drug Delivery Reviews, 47 (2001) 113-131.

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Highlights



A novel thermoresponsive nanomicelles based on star-liked block copolymers was synthesized via ring-opening (ROP) and reversible addition–fragmentation chain transfer (RAFT) polymerization.



The biodegradable star-like polymeric micelle was developed to overcome the limitations of docetaxel (DTX)-loading as a hydrophobic anticancer drug.



The release behaviors of DTX from the star-liked polymer exhibited that the rate of DTX release could be efficiently controlled by temperature and pH value.



DTX-nanomicelles can be used as an anticancer drug delivery system, due to its useful biocompatibility and excellent physicochemical properties.

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Graphical abstract

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