Synthesis of hyaluronic acid-based polymersomes for doxorubicin delivery to metastatic breast cancer

Journal Pre-proofs Synthesis of hyaluronic acid-based polymersomes for doxorubicin delivery to metastatic breast cancer Mahsa Shahriari, Seyed Mohammad Taghdisi, Khalil Abnous, Mohammad Ramezani, Mona Alibolandi PII: DOI: Reference:

S0378-5173(19)30880-4 https://doi.org/10.1016/j.ijpharm.2019.118835 IJP 118835

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

2 August 2019 22 October 2019 28 October 2019

Please cite this article as: M. Shahriari, S. Mohammad Taghdisi, K. Abnous, M. Ramezani, M. Alibolandi, Synthesis of hyaluronic acid-based polymersomes for doxorubicin delivery to metastatic breast cancer, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm.2019.118835

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Synthesis of hyaluronic acid-based polymersomes for doxorubicin delivery to metastatic breast cancer Mahsa Shahriari1, 2, Seyed Mohammad Taghdisi3, Khalil Abnous1,4, Mohammad Ramezani 1, 2*, Mona Alibolandi1, 2* 1Pharmaceutical

Research Center, Pharmaceutical Technology Institute, Mashhad University of

Medical Sciences, Mashhad, Iran 2Department

of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical

Sciences, Mashhad, Iran 3Targeted

Drug Delivery Research Center, Pharmaceutical Technology Institute, Mashhad University

of Medical Sciences, Mashhad, Iran 4Department

of Medicinal Chemistry, School of Pharmacy, Mashhad University of Medical Sciences,

Mashhad, Iran

Corresponding Authors: Dr. Mona Alibolandi, Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Tel/Fax: +98 51 38823255, E-mail: [email protected] Prof. Mohammad Ramezani, Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Tel/Fax: +98 51 37112470, E-mail: [email protected]

1|Page

Abstract In the current research, the synthesis of polysaccharide-based polymersomes for targeted delivery of doxorubicin is reported. To this aim, doxorubicin was encapsulated in aqueous compartment of hyaluronan-polycaprolactone polymersomes via nanoprecipitation method. Then the therapeutic index of the prepared formulation was studied in metastatic breast cancer model in vitro and in vivo. The size of obtained polymersomes was 146.2 ± 9.6 nm and offered the efficiency of encapsulation and the content of loading %54.9 ±4.0 and %3.6 ±0.4, respectively. The obtained results exhibited that the HA-PCL polymersomes controlled the release of DOX in a sustained manner. Then, the CD44-receptor mediated endocytosis through hyaluronan shell of the prepared formulation was confirmed in murine 4T1 and human MCF-7 cancer cell lines implementing flow cytometry and MTT assays. Much better in vivo antitumor efficacy and wider tumor tissue necrosis and better bio-distribution features of this formulation in comparison with PEG-PCL-DOX nanoparticles suggested that HA-PCL-DOX can potentially reduce off-target effects due to its targeting capability.

Keywords: Nanopolymersome, Doxorubicin, Hyaluronic acid, Breast cancer, 4T1, Polycaprolactone

2|Page

1. Introduction Breast cancer is one of the most commonly diagnosed cancers and the second cause of cancer related death among women globally (Nagini, 2017). Various strategies have been implemented to control the disease in different breast cancer stages such as chemotherapy, endocrine therapy, surgery and radiation therapy (Cazzaniga et al., 2017). Undesirable adverse effects may occur after chemotherapy. It has been demonstrated that doxorubicin (DOX), a member of anthracycline family, is a chemotherapy drug using for treatment of lung, breast, brain, prostate ,cervix and ovarian carcinomas, alone or combined with other anticancer drugs (Kumar et al., 2014). DOX is considered as one of the first line treatments in breast cancer (Zhang et al., 2019). Meanwhile, clinical uses of DOX have been limited because of dose-dependent irreversible cardiotoxicity. Also, the other obstacle that restrict clinical administration of DOX is the acquisition of drug resistance (Chen et al., 2018; Franco et al., 2018). To overcome these hurdles, more effective delivery of DOX to tumor sites is required. Recent developments in nanotechnology have proposed novel therapeutic platforms to deliver their cargo selectively to cancer cells while minimizing systemic toxicity in order to overcome most important limitation in cancer therapy. Numerous nanoparticles are under investigation to carry multiple agents and control their release in the site of action, along with their high ligand density because of high surface/volume ratio (Jahangirian et al., 2019; Shafei et al., 2017). Nanoscale platforms increase the efficacy of anti-tumor molecules because of the enhanced permeation and retention effect which is resulted from leakiness of tumor blood vessels and reduced lymphatic vessels flow in tumor tissues (Kalyane et al., 2019; Tahmasbi Rad et al., 2019; Zhao et al., 2019). Various types of carriers can be implemented for drug delivery including lipid or polymeric nanoparticles, nano-emulsions, liposomes, polymersomes, micelles, exosomes, nanogels, quantum dots and dendrimers (Li et al., 2017). Liposomes as drug carriers have already found their way into clinical use, because of their special features comprising biocompatibility and colloidal stability. On the other hand, PEGylation of liposomal formulations can enhance their circulation time in blood (Müller and Landfester, 2015). It has been shown that DOXIL® (liposomal form of DOX) administration improved survival rate in comparison with conventional DOX in cancerous patients (Ngan and Gupta, 2016). The structures of natural liposomes inspire construction of fully synthetic polymersomes with advantages over liposomes such as longer circulation times, mechanical robustness, a wide range of modification-possibilities, and enhanced stability. 3|Page

These polymeric vesicles with amphiphilic bilayers provide encapsulation of large quantities of both hydrophilic and lipophilic agents (Meng et al., 2009). In this regard, Alibolandi et al. fabricated folate-targeted dextran-poly (lactic-co-glycolide) based polymersomes containing docetaxel. This formulation exhibited sustained release of DTX, with enhanced uptake of anticancer agent, along with more toxicity in MCF-7 and 4T1 cell lines. Owing to the folate antenna, superior tumor-targeting and improved therapeutic index were observed (Alibolandi et al., 2016). A cluster of differentiation-44 (CD44) is a transmembrane glycoprotein overexpressed in several solid tumors, such as breast and gastric cancer, hepatocarcinoma, and melanoma (Lim et al., 2011). Therefore, many studies have been conducted to improve drug delivery performance and endocytotic uptake in cancerous cells through targeting overexpressed CD44 receptors on cancerous cells surfaces (Jang et al., 2013; Lee et al., 2018). Hyaluronic acid (HA), known as a hydrophilic polymer, can be used in drug delivery systems due to its good biocompatibility and bioavailability, also targeting capability of CD44 receptors overexpressed in various cancer cells. Tumor onset and progression are the most important events which are regulated by interaction of HA with CD44 (Karousou et al., 2017). In this regard, Jiang et al. developed HA-modified laponite® (LAP) nano-disks for the entrapment of DOX and its specific delivery to the cancer cells which overexpress CD44 receptors. This pH-sensitive nanoplatform demonstrated high loading efficiency with enhanced anti-tumor efficacy and improved uptake through CD44-mediated endocytosis (Jiang et al., 2019). In this regard, current study was performed to develop novel hyaluronic acid-poly caprolactone (HA-PCL) nanopolymersomes. Doxorubicin was encapsulated in the aqueous compartment of nanopolymersomes via nanoprecipitation procedure for preparation of the sustained release of DOX for cancer therapy. Afterwards, cytotoxicity effect and cellular uptake were evaluated in MCF-7 and 4T1 cell lines. Moreover, the therapeutic index and biodistribution of DOX-loaded HA-PCL polymersomes were evaluated in vivo on 4T1 metastatic mouse breast cancer model. 1.1.

Hypothesis

We hypothesize that hyaluronan-based polymersomes with efficient loading content of DOX are prepared by self-assembly of hyaluronan-polycaprolactone amphiphilic diblock copolymer. Besides, we assume that the hyaluronan shell of polymersomes would result in particular delivery of chemotherapeutic agents to CD44 overexpressing breast cancer cells with higher anticancer agent accumulation and better cytotoxic effects in vitro and in vivo.

4|Page

2. Materials and methods 2.1.

Materials and cell lines

Hyaluronic acid (HA; average Mw: 5,000 Da), polycaprolactone (PCL; average Mw: 14,000 Da),

1,2-ethylenediamine,

N-hydroxysulfosuccinimide

(NHS),

1-ethyl

3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDC), sodium cyanoborohydride, succinic anhydride, 4-dimethylaminopyridine (DMAP), trimethylamine, and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma– Aldrich (Schnelldor, Germany). Doxorubicin hydrochloride (DOX) was obtained from Euroasia Co., Ltd. (Delhi, India). Roswell Park Memorial Institute (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin–streptomycin, and trypsin were obtained form from GIBCO (Darmstadt, Germany). All of the other solvent and chemicals and reagent grade solvents used in this research were obtained from Merck & Co (Darmstadt, Germany) without additional purification. Human breast adenocarcinoma (MCF-7), and Murine mammary carcinoma (4T1) cell lines, were prepared from Iran Pasteur Institute. RPMI-1640 medium is used for the culture of both cell lines which properly supplemented with 1% antibiotics (100 units/ml of penicillin and 100 µg/ml streptomycin) and 10% of fetal bovine serum (FBS) at 37 oC in a moisturized atmosphere containing 5% CO2. 2.2.

Synthesis and characterization of HA-PCL copolymer

2.2.1. Characterization 1H

NMR (proton nuclear magnetic resonance) spectroscopy of synthesized polymers in each

step were obtained in deuterated solvents recorded on a Bruker Avance III 300 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). Infrared spectra (FTIR) of the synthesized polymers were obtained as follows: Polymers (10 mg) were added to 100 mg of KBr to provide a KBr disk. Then, it was grinded for 3-5 min. The die was put together with the powder into the QwikHandi-Press to produce a tablet. The prepared tablets were placed in the spectrometer Perkin-Elmer Model 1000 to record FTIR spectra. 2.2.2. Reducing-end amination of hyaluronic acid Hyaluronic acid solution (Mw= 5000 g/mol, 10 mg/ml) was added to a round-bottom flask, then 1,2-ethylenediamine solution (in PBS buffer with 10 equivalent concentration of the HA) was drop-wisely added to the solution of HA. Then, the mixture was reacted for 4 hours. Subsequently, sodium cyanoborohydride was added to the mixture in an ice-bath and continuously stirring of the final reaction mixture was allowed to carry out at ambient 5|Page

temperature for 18 h (Liu et al., 2010). Next day, the obtained solution was evaporated by rotary evaporator to eliminate excess diamine and water. Thereafter, the crude product was dialyzed against PBS buffer at 4 °C for 48 hours. Afterwards, the purified polymer was freeze dried in order to prepare amine-functionalized HA. The 1H NMR spectrum of the HA-NH2 in D2O was recorded at room temperature to verify amine functionalization of HA. 2.2.3. Synthesis of carboxylated PCL Firstly, PCL (Mw=14000 g/mol) was dissolved in 1,4 dioxane (50.4 mg/ml) and then succinic anhydride (1.2 equivalent of PCL), DMAP (1.2 equivalent of PCL), and triethylamine (1.2 equivalent of PCL) were added into the PCL solution and allowed to stir for 24 h. Then, the solvent was evaporated by rotary evaporator and hydrolysis using hot water was performed. Finally, the solution was extracted three times with dichloromethane and vacuum dried (Zhang et al., 2014). Then the polycaprolactone was activated by EDC/NHS. In this regard, polycaprolactone (0.025 mmol) and EDC (4 equiv.) and NHS (4 equiv.) were dissolved in dichloromethane, and then stirred at ambient temperature for 24 h (Liu et al., 2010). Then dichloromethane was evaporated and the crude product was washed with methanol, freezedried and maintained at -20 °C for further use. The 1H NMR spectrum of the PCL-COOH was taken in deuterated chloroform (CDCl3) at ambient temperature. 2.2.4. Coupling reaction of aminated hyaluronic acid to carboxylated polycaprolactone The aminated hyaluronic acid (1.2 equivalent of activated NHS-PCL) was dissolved in 10 ml dimethylformamide (DMF) and then N, N-diisopropylethylamine was added at 100 °C and stirred for 24 h. After that, the solvent was eliminated using rotary evaporator. The final product was dialyzed (Spectra/Por 6 Pre-wetted Standard Dialysis Tubing, 6-8 kD MWCO) against distilled water for two days for removing unreacted HA, and then lyophilized by freeze-drying for 24 h and kept at −20 °C for further use. The structure of final product was verified using 1H

NMR spectroscopy in DMSO-d6. The existence of amide linkages in HA-PCL was featured

implementing Fourier transform infrared (FT-IR) spectrophotometer (Paragon 1000, PerkinElmer, USA). For this reason, a solution of HA-PCL copolymer in chloroform was prepared in order to construct the polymeric thin layer on the horizontal area of the NaCl crystal. A Differential Scanning Calorimeter (DSC, Mettler Toledo DSC 822, Schwerzenbach, Switzerland) equipped with liquid nitrogen cooling accessories was implemented to monitor 6|Page

thermal transitions of HA-PCL, HA and PCL. The samples (2 mg) were heated from 20 to 250 ℃ at a heating rate of 10 ℃/min.

7|Page

OH OH

A

ONa O

HO O

O

O

O

NH

O

O

O

H N

OH

HO

CH3

CH3

n

NH2

NH

O

O

n

O CH3

Hyaluronic acid-amino

Hyaluronic acid

O

O

O

B

OH

O

OH O

HO O

NaBH3CN

OH

ONa

NH2

H2N

O

HO

NH

OH

O

O

O

Succinic anhydride

O

DMAP n

,

O OH

Triethylamine

n

N

Polycaprolactone terminated with carboxyl group

N

Polycaprolactone

O

N

OH OH OH

C

HO O

ONa O

O

O HO

OH

O O

OH O OH

O

H N

OH NH

CH3 n

O

+

O

EDC/NHS

O OH

NH2

nO

HO O

ONa O

O HO

O

OH

O O

OH O

OH NH

CH3 n

O

O

H N

O N H

O

O CH3

CH3

Hyaluronic acid-amino

Polycaprolactone terminated with carboxyl group

Hyaluronic acid-polycaprolactone

Figure 1. Synthesis of aminated hyaluronic acid (A); Synthesis of carboxylated polycaprolactone (B); Coupling reaction of aminated hayluronic acid to carboxylated PCL.

8|Page

n

2.5.

Polyethylene glycol-polycaprolactone copolymer synthesis

To synthesize the copolymer, PEG 5000 (2 g) was placed into a flask in a Milestone Microsynth microwave (Milestone, Italy) at 1200 W and 130 °C for 10 min to dry PEG. In the next step, caprolactone (5 g) and 20 µL of Tin(II) 2-ethylhexanoate was added to the dried PEG, and then stirred and irradiated at 120 oC and 1000 W under 50 rpm for 120 min. After that, to purify the di-block copolymer, the final product was dissolved in chloroform and then precipitated by diethyl ether. The excess solvent was evaporated using rotary evaporator at 30 oC for 30 min. To remove the excess water, the products were lyophilized using freeze dryer and stored at – 20 °C until further use. The 1H NMR spectrum of the PEG-PCL di-block was recorded in CDCl3 at room temperature. Agilent GPC-Addon apparatus with Plgel® columns was used to calculate the molecular weights and polydispersity of obtained di-block copolymer. Tetrahydrofuran as an eluent and polystyrene standards as a calibration were used. 2.6.

Preparation of HA-PCL and HA-PCL-DOX nanoparticles

Blank HA-PCL NPs and doxorubicin-loaded HA-PCL polymersomes were prepared using the nanoprecipitation method. For this purpose, HA-PCL copolymer (7.5 mg) was dissolved in 1.5 ml dimethylsulfoxide (DMSO), subjected to sonication for 30 minutes in 60 ºC, while doxorubicin was dissolved in 3 ml deionized water, separately. The organic phase was added dropwise to the hydrophilic phase under probe-sonication. Then, the solution was allowed to stir for 2 h at 60 ºC and dialyzed (Spectra/Por 6 Pre-wetted Standard Dialysis Tubing, 6-8 kD MWCO) against 2 L deionized water for 6 h for elimination of solvent and free doxorubicin. The final formulation was freeze-dried. The calculation of doxorubicin content of polymersomes was performed using the provided standard curve of DOX by UV spectrophotometer at 480 nm. Briefly, after dissolving of the DOX-encapsulated nanoparticles in DMSO, the absorbance of released free drug was measured at 480 nm. The drug loading content (LC) and encapsulation efficiency (EE) of nanoparticles were calculated with the following Equation 1 and 2, respectively. EE% = Amount of the entrapped drug in the formulation / Amount of used drug in initial stage (1) LC% = Amount of drug in the nanoparticles / Amount of nanoparticles

9|Page

(2)

2.7.

Preparation of PEG-PCL-DOX nanoparticles

DOX-loaded PEG-PCL NPs were prepared via double emulsion method. In this regard, PEGPCL copolymer was dissolved in dichloromethane/acetone mixture (4/1, v/v) and emulsified with aqueous solution of doxorubicin. In the next step, the emulsified solution was added dropwise to 4 ml of PVA (5%, v/v) in the period of 10 min during sonication. Afterwards, the prepared emulsion was added to 10 ml of PVA (0.1%, v/v) slowly while stirring (rpm 800). For elimination of dichloromethane/acetone, stirring was continued for 16 h. Finally, the NPs were purified using centrifugation at 15,000 g for 30 min at ambient temperature and in the final step, they were washed twice with distilled water for elimination of PVA. 2.8.

Particle size distribution and zeta potential measurements

A suspension of nanoparticles was prepared in deionized water (1mg/1ml) and the measurements of particle size distribution, polydispersity index and the charge of nanoparticle surface (zeta potential, mV) were measured through a DLS (Dynamic Light Scattering) technique (Nanopartica SZ-100; HORIBA Ltd, Kyoto, Japan). 2.9.

Morphological features of the prepared formulations

Field emission scanning electron microscope (FESEM, TESCAN BRNO- Mira3 LMU, Czech Republic) was applied to evaluate the particle size and morphological characteristics of the HA-PCL-DOX and PEG-PCL-DOX NPs. The self-assembly properties of the synthesized PEG-PCL and HA-PCL copolymers were investigated using Atomic Forced Microscopy (AFM). An atomic force microscope (AFM, Nano Wizard II; JPK Instruments, Berlin, Germany) analysis under AFM tapping mode was applied to demonstrate the vesicular properties of the PEG-PCL and HA-PCL copolymers after self-assembly process in a partially dehydrated state in air. The sample was prepared as follows: polymersomes were diluted and dispensed onto the AFM mica disc and dried for 24 h, at ambient temperature. 2.10.

In vitro release of drug

For in vitro measurements of DOX release from the prepared formulation, membrane-diffusion method was used. For this purpose, 1 ml of suspended form of all formulations (HA-PCL-DOX and PEG-PCL-DOX) were placed into a dialysis sac with MWCO of 3500 Da, then submerged in either 20 mL citrate buffer (pH 5.5) or phosphate-buffered saline (PBS, pH 7.4) and 10 | P a g e

maintained in a shaking incubator at 37 °C and 80 rpm. At predetermined time points, 1 mL of each formulation were withdrawn and 1 mL of fresh buffer was replaced. To determine the DOX content of all samples UV-Visible spectrophotometer (Cary 100 BIO UV–Vis spectrophotometer, Varian, CA, USA) at 480 nm was implemented. All experiments were accomplished in triplicate. Then, the percentage cumulative DOX release from the prepared nanoparticles were calculated up to 250 h. 2.11.

Cell viability assays

4T1 and MCF-7 cell lines were cultured in RPMI medium, supplemented with 10% (v/v) fetal bovine serum (FBS), 1% penicillin–streptomycin at 37 °C, and 5% CO2. In the first step, 4T1 and MCF-7 cell lines were seeded into 96-well plates at a density of 5 × 103 cells per well and then incubated overnight at 37˚C. After 6 h of treatment with either HA-PCL-DOX or free DOX (concentrations, 0.78–50 μg/mL for 4T1 and 0.47-30 μg/mL for MCF-7 cell lines), the media were replaced with fresh complete media, following incubation for 48 h at 37 °C in a humidified incubator. Next, adding 20 μL of 5 mg/mL MTT solution to each well was performed and then incubated for a further 4 h in a humidified incubator. In the final step, media were aspirated and DMSO (100 μL) was added to each well. The absorbance measurement was accomplished at 570 nm with reference wavelength of 630 nm using an Infinite® 200 PRO multimode microplate reader (Tecan Group Ltd., Männedorf, Switzerland). To verify the selective targeting of CD44 receptors in the 4T1 and MCF-7 cells by HA-PCLDOX NPs; a competitive experiment was carried out. In this experiment, excess amounts of free hyaluronic acid (0.5 mg/ml) were added to each well 30 minutes before adding the targeted formulation (HA-PCL-DOX). 2.12.

Cellular uptake

For cellular uptake investigation of targeted (HA-PCL-DOX) and non-targeted (PEG-PCLDOX) formulations, BD FACSCalibur™ Flow Cytometer (BD Biosciences, San José, CA, USA) equipped with a 488nm argon-ion laser in the FL2 channel was used. 4T1 and MCF7 cell lines (4×104 cells/well) were seeded into 12-well plates. After 24 h, either HA-PCL-DOX, PEG-PCL-DOX or free DOX (DOX equivalent concentration 3 µg/ml) were added to wells. Media was removed 2 h post-treatment and cells were trypsinized. The cells suspension was centrifuged at 1400 rpm for 7 min at 4 °C. Afterwards, the cell pallet was washed three times with cold PBS pH 7.4 (twice in 1 ml PBS). Then cell pellets were dissolved in cold PBS (200 µL) for flow cytometry analysis. All data were analyzed by FlowJo 7.6 software. 11 | P a g e

To confirm the selective targeting of CD44 receptors presented on the 4T1 and MCF-7 cell lines by HA-PCL-DOX NPs, a parallel experiment was performed using free hyaluronic acid (0.5 mg/ml) for each well 30 min prior to adding the targeted formulation. 2.13.

Ex vivo imaging

For ex vivo imaging, female BALB/c mice were inoculated with 4 × 105 4T1 cells in 80 µL PBS per mouse subcutaneously. After reaching tumor size to 100 mm3, they were divided into four groups (control, free DOX, HA-PCL-DOX, PEG-PCL-DOX) and the intravenous injection with 100 μL (with 5 mg/kg dox equivalent) via tail vein was administrated. After 12, 24 h post injections, cervical dislocation was chosen to euthanize mice. Then the kidneys, lungs, spleen, liver, heart, and tumor were collected for fluorescent imaging implementing KODAK IS in vivo imaging system. KODAK Molecular Imaging® software 5.0 was implemented to evaluate the biodistribution of free DOX, HA-PCL-DOX and PEG-PCL-DOX in 4T1-tumor bearing mic using a fluorescence intensity quantification assay. 2.14.

In vivo therapeutic efficacy

All animal studies were done in accordance and prior approval of Institutional Animal Care and Use Committee of the School of Pharmacy (Mashhad University of Medical Sciences). For the in vivo evaluation, four to six-week old (18-20 gram) female BALB/c mice were purchased from Pasteur Institute in Iran (Tehran, Iran) and maintained under conventional conditions in the laboratory animal care facility. Then, we established mouse tumor model by local subcutaneous injection of 4T1-tumor cells (4 × 105 cells/ in 80 µL PBS solution) into the right flank of each mouse. After 8 days of injection, mice were distributed into four groups (n=5), control, free DOX, PEG-PCL-DOX and HA-PCL-DOX, and they received 100 µL of either free DOX, HA-PCL-DOX or PEG-PCL-DOX (DOX equivalent: 5 mg/kg) via a single tail intravenous administration. For negative control group, the injection of NaCl solution (0.9% w/v) was performed. The calculation of tumor volume for each mouse was made after measurement of following parameters: largest diameter, smallest diameter, as well as the depth of the tumor. After that, we entered these parameters into a × b × w/2 equation where “a” and “b” are largest and smallest diameters of tumor, respectively, and “w” is its depth. Moreover, the toxicity assessment of free DOX, HA-PCL-DOX, and PEG-PCL-DOX NPs was performed by measurement of the body weight and survival rates. Also, we followed up all groups of mice for 30 days after intravenous injection or when one of the below situations for euthanasia was occurred as follows: (1) reduction of mice body weight was observed below 20% of their pre12 | P a g e

study weight; (2) one of the tumor diameters was more than 200 mm; (3) mice showed any sign of illness or become unable to feed; or (4) they died unexpectedly. 2.15.

Systemic toxicity: pathological evaluation

Animals were euthanized 30 days after the intravenous administration of free DOX, HA-PCLDOX NPs and PEG-PCL-DOX NPs (n=5, 5 mg/kg DOX equivalent). After euthanasia of all mice, the kidneys, lungs, spleen, liver, heart, and tumor were collected, following three times washing with PBS and fixed in a 10% formalin solution. Tissues that have been formalin-fixed and paraffin-embedded were sectioned (5 μm diameter) and staining procedure with hematoxylin and eosin (H&E) was used. The images were prepared at 10× and 40× magnification. 2.16.

Statistical analysis

To carry out statistical data analysis, we used a one-way analysis of variance (ANOVA). A probability value of 0.05 (p-value=0.05) was considered as the cut-off for significance that means if the probability value is 0.05 or lower, that suggests no difference between the means. Results are indicated as mean ± standard error of mean (S.E.M). 3. Results and discussion 3.1.

Synthesis and characterization of HA-PCL copolymer

The amphiphilic diblock copolymer of HA-PCL comprising HA5000 and PCL15000 was successfully synthesized by three step reactions. In the first step, the reductive amination of HA was performed followed by carboxylation of PCL. Then EDC/NHS reaction was implemented to couple the aminated HA (NH2-HA) to carboxylated PCL (COOH-PCL) as shown in Fig. 1. Fig. 2A shows the 1H NMR spectrum of HA and EDA–HA conjugate. The characteristic chemical shifts corresponding to HA are observed at 2.0 and 3.3–4.7 ppm. The resonance peak at 2.92 ppm is corresponded to the CH2 of EDA which conjugated to the terminal of HA, demonstrating efficacious reductive amination of HA. Fig. 2B illustrates the 1H NMR spectra of PCL and carboxylated PCL (COOH-PCL). The hydrogen resonance at 4.1 ppm is assigned to CH2 of the caprolactone next to the C=O while other hydrogen resonances at 2.3 and 1.6 ppm correspond to CH2 residues of the PCL appeared as multiplet. The small peak at 11-12 ppm was assigned to the hydrogen of terminal carboxyl

13 | P a g e

group of COOH-PCL polymer which confirmed the successful conversion of PCL to COOHPCL. Finally, Fig. 2C demonstrates the 1H NMR spectrum of HA-PCL in which peaks corresponding to either the PCL and HA segments appeared at 4.8, 5.2, 1.50 ppm and 2, 3.3 ppm, respectively. Conjugation of NH2-HA to COOH-PCL was ascertained by FT-IR analysis by appearance of the amide bond peak at 1626 cm-1 (carbonyl, C=O) in HA-PCL copolymer, which was absent in COOH-PCL and HA polymers (Fig. 3A). The DSC thermogram of the HA-PCL copolymer demonstrated two wide endothermic peaks at 44-68°C, 120–165 °C while a single sharp peak at 60 °C and a broad peak at ~150–190 °C were recorded in the thermograms of PCL and HA, respectively. The aforementioned results verified the formation of HA-PCL copolymer. In this regard, after coupling of HA to PCL, the melting endothermic peaks of HA and PCL segments in HA-PCL copolymer were shifted to lower temperatures with wider ranges (Fig. 3B). These results confirmed the conjugation of PCL to HA and preparation of an amorphous diblock copolymer.

14 | P a g e

A

B

C

HA-PCL

Figure 2. 1H NMR spectra of hyaluronic acid and aminated hyaluronic acid (A); polycaprolactone and carboxylated polycaprolactone (B); hyaluronic acid-polycaprolactone diblock copolymer.

15 | P a g e

A

B

Figure 3. (A) FTIR spectrum of aminated hyaluronic acid, carboxylated-polycaprolactone and hyaluronic acid-polycaprolactone diblock copolymer. (B) Differential scanning calorimetry (DSC) thermogram of hyaluronic acid, polycaprolactone and hyaluronic acid-polycaprolactone diblock copolymer.

3.2.

Synthesis and characterization of PEG-PCL copolymer

Microwave-assisted ring opening polymerization (MROP) method was utilized for amphiphilic PEG-PCL diblock copolymer synthesis. Fig. 4 illustrates the 1H NMR spectrum of PEG-PCL. The characteristic CH2 of PEG block is assigned to peak appeared at 3.6 ppm and other characteristic peaks of PCL were also observed in the 1HNMR spectrum.

16 | P a g e

GPC chromatogram of the di-block PEG5000-PCL15000 demonstrated MW 16228 and Mn 9381 with PDI of 1.72.

Figure 4. 1HNMR spectrum of PEG-PCL copolymer.

3.3.

Encapsulation efficiency and characterization of nanoparticles

Doxorubicin encapsulated nanopolymersomes based on HA-PCL copolymer (HA-PCL-DOX), was prepared through nanoprecipitation technique. Based on our results, DOX was encapsulated in HA-PCL polymersomes with encapsulation efficiency (EE) and loading content (LC) of %54.9±4.0 and %3.6±0.4, respectively. DOX encapsulation in PEG-PCL copolymer was performed implementing double emulsion method with EE and LC of %26 ± 2.5 and 1.3%±0.2, respectively. Size distribution of polymersomes was determined to be 146.2±10.0 nm (polydispersity index =0.12 ± 0.03, zeta potential=-42.1 ± 0.3, n=2) for HA-PCL-DOX and 106±21 nm (polydispersity index=0.2±0.02, zeta potential=-9.2±0.4) for PEG-PCL-DOX. The diameters of either HA-PCL-DOX or PEG-PCL-DOX are less than 200 nm, considering that particles smaller than 200 nm are capable of being accumulated at the tumor site via EPR effect thereby reducing the systemic adverse effects and improving the pharmacokinetics of entrapped drugs. Furthermore, high negative value of zeta potential is crucial for nanoparticle stability. Owing to the presence of hyaluronic acid on the HA-PCL nanopolymersome surfaces, the resultant HA-PCL-DOX NPs has a high negative charge.

17 | P a g e

3.4.

Morphological evaluation of HA-PCL self-assembled structure

Scanning electron microscopy (SEM) was utilized to evaluate the structural properties of selfassembled nanoparticles. Fig. 5A&B demonstrates SEM image of the HA-PCL-DOX and PEG-PCL-DOX NPs, demonstrating spherical nanoparticle with average size of 140 and 110 nm for HA-PCL-DOX and PEG-PCL-DOX, respectively. HA-PCL-DOX and PEG-PCL-DOX structures in a dehydrated state were further evaluated implementing atomic force microscopy (AFM). The self-assembled polymersomes showed ‘‘donutlike’’ structures with thinner centers compared with their edges (Fig. 5). This structure supported the idea that the spherical unimodal NPs in SEM images are waterfilled vesicular self-assembled structures that would collapse into dehydrated “donutlike” morphology in AFM profile.

18 | P a g e

A

B

C

D

E

F

Figure 5. SEM images of HA-PCL-DOX (A) and PEG-PCL-DOX (B). AFM images of PEG-PCLDOX (C); HA-PCL-DOX (D). Height profile of HA-PCL-DOX (E) and PEG-PCL-DOX (F).

3.5.

In vitro drug release profile of HA-PCL-DOX nanopolymersomes

In the next step, the in vitro release patterns of anticancer agent (DOX) from HA-PCL-DOX and PEG-PCL-DOX nanoparticles in either citrate buffer (pH=5.5) or phosphate-buffered saline (PBS, pH=7.4) were examined (Fig. 6). 19 | P a g e

The obtained results from release profile demonstrated the higher rate of DOX release at pH 5.5 in comparison with pH 7.4 for both systems (HA-PCL-DOX and PEG-PCL-DOX). The release rates of DOX accelerated in pH 5.5 because of the increased water solubility of protonated DOX in citrated buffer (pH 5.5). Moreover, the HA-PCL-DOX particulate system illustrated lower DOX release in either PBS or citrate buffer in comparison with PEG-PCLDOX demonstrating the capability of the HA-PCL polymersome in to provide better controlled release of DOX in a sustained manner. On the other hand, the aforementioned low rate of release in HA-PCL polymersome system may be ascribed to the electrostatically interaction between positively-charged DOX and negatively-charged HA.

A

B

Release Profile (HA-PCL-DOX)

80

pH=5.4 pH=7.4

60

40

20

0 0

50

100

150

200

250

Cumulative DOX release(%)

Cumulative DOX Release %

80

Release Profile (PEG-PCL-DOX) pH=5.4 pH=7.4

60

40

20

0

Time (h)

0

50

100

150

200

250

Time(h)

Figure 6. Release profile of HA-PCL-DOX (A) and B:PEG-PCL-DOX (B) in PBS, pH 7.4 and citrate buffer, pH 5.5. (n = 3, error bars represent standard error of the mean).

3.6.

Cellular uptake study by flow cytometry

In order to determine in vitro cellular uptake and cancer cell targeting of HA-PCL-DOX NPs against the CD44 overexpressing cells (MCF-7 and 4T1), we compared the cellular uptake of HA-PCL-DOX, PEG-PCL-DOX and free DOX after 2 h incubation implementing flow cytometry analysis (Fig. 7A&B). As illustrated in the Fig. 7A&B, for both cell lines (4T1 and MCF7), cellular uptake of HAPCL-DOX was higher than that of PEG-PCL-DOX and free DOX. Although, a significant reduction in uptake of HA-PCL-DOX by cells treated with free HA was observed before addition of HA-PCL-DOX in comparison with HA-PCL-DOX treated cells without free HA addition as competing ligand. 20 | P a g e

The obtained results confirmed the higher uptake of HA-PCL-DOX when compared with PEGPCL-DOX and free DOX in both cell lines (4T1 and MCF-7) which was attributed to endocytosis mediated by CD44 receptor. However, the cellular uptake of non-targeted nanoparticles (PEG-PCL-DOX) was less than that of free DOX or HA-PCL-DOX due to the over-PEGylation of the nanoparticles surfaces. Various researches have investigated the uptake of nanoparticulate systems by endocytosis mediated by CD44 receptor. In this regard, Jeannot et al. demonstrated the hyaluronan-based nanoparticles which has ability to target CD44 receptors of lung cancer in vitro and in vivo. The prepared system illustrated active targeting of CD44-overexpressing tumors. They exhibited that using CD44 targeted nanoparticles increased DOX uptake by the CD44+ cancer cells overexpressing CD44 receptor (Jeannot et al., 2016). 3.7.

MTT assay

The cellular uptake experiment confirmed the augmented cellular uptake of HA-PCL-DOX in CD44-overexpressing tumor cells. To investigate the aforementioned issue, in vitro cytotoxicity of free DOX and HA-PCL-DOX NPs in two different groups with or without pretreatment with hyaluronic acid (HA) before addition of nanoparticles was evaluated in two cell lines (4T1 and MCF-7). The IC50 of HA-PCL-DOX was 3.2 µg/mL and 0.4 µg/mL in 4T1 (Fig. 7C) and MCF-7 (Fig. 7D) cells respectively, while free DOX at the identical concentration, exhibited 37% and 40% survival percent in 4T1 and MCF-7, respectively. Although the cellular uptake of HA-PCLDOX was higher than free DOX as demonstrated by flow cytometry analysis, the lower cellular cytotoxicity of HA-PCL-DOX compared to free DOX was observed because of the bilayer stability of the HA-PCL polymersome and its controlled-release properties. Hyaluronic shell increased the cellular uptake of the polymersomes into cancer cell lines which overexpressed CD44 receptor (MCF-7 and 4T1) while demonstrating inefficiency towards the HA-pretreated cells. This is in agreement with the MTT results exhibited in Fig. 7C, D, in which HA treatment prior to HA-PCL-DOX exposure significantly reduced cellular toxicity of HA-PCL-DOX toward 4T1 and MCF-7 cell lines. The obtained results suggest that HA shell may selectively increase the transportation of DOX to both cells (MCF-7 and 4T1) due to CD44 mediated endocytosis. We believe that the obtained polysaccharide-based polymersome formulation encapsulating DOX can be used as a versatile nanomedicine for breast adenocarcinoma treatment.

21 | P a g e

C

80

***

Untreated by HA ***

***

Free Dox

60 Cell Viability (%)

60

40

20

*

****

**

**

*

40

20

00

6.

25

0

. 12

50

0

. 25

0 00

. 50

00

0

Concentration (µg/ml)

0 30 .0 00

2 3.

15 .0 00

0

7. 50 0

56

3. 75 0

1.

1. 87 5

0.

0 70

0. 93 8

Cell Viability (%)

Pretreated by HA Untreated by HA Free Dox

Prereated by HA **

0

MCF-7

0. 46 9

80

D

4T1

Concentration of DOX(µg/ml)

Figure 7. Flow cytometry analysis of (A) 4T1 and (B) MCF-7 cell lines after 2 h incubation with free DOX, PEG-PCL-DOX and HA-PCL-DOX with or without pretreatment with hyaluronic acid. Cytotoxicity of free DOX and HA-PCL-DOX NPs after 48 h incubation of 4T1 (C) and MCF-7 (D) at 37°C (n = 4, error bars represent standard error of the mean).

3.8.

Ex vivo DOX florescence imaging

To compare the relative concentration of anticancer agent within substantial organs and tumor tissue, free DOX, HA-PCL-DOX and PEG-PCL-DOX NPs was applied to inject in 4T1 tumor bearing female BALB/c mice for evaluation of DOX fluorescence. After intravenous injection of mice via tail vein with 100 µL of either free DOX, PEG-PCLDOX or HA-PCL-DOX (equivalent DOX concentration: 5 mg/kg), they were sacrificed at 12 and 24 h after injection and major organs (kidneys, heart, spleen, lung, liver and tumor) were removed and washed with PBS buffer. The fluorescence intensity of DOX in organs were imaged as shown in Fig. 8.

22 | P a g e

The obtained results exhibited that the tumor accumulation of HA-PCL-DOX treated mice were significantly superior than that of PEG-PCL-DOX treated mice 12 h post-injection which could be attributed to the CD44 receptor targeting capability of the hyaluronic block in the HAPCL-DOX polymersomal formulation. Furthermore, the tumor accumulation 24 h postinjection was slightly higher in mice injected by PEG-PCL-DOX when compared with those treated with HA-PCL-DOX which confirms passive targeting of PEG-PCL-DOX polymersomal formulation through EPR. Our data are inconsistent with previous studies. In this regard, Wu and coworkers demonstrated the faster and higher accumulation of peptide-targeted liposomes in tumor site at 24 h postinjection in comparison with non-targeted system which was gradually accumulated at tumor site via EPR up to 48 h post-injection (Wu et al., 2018). On the other hand, the uptake of both formulations in most non-malignant tissues including kidney, heart, spleen and lung was identical except liver which was significantly lower in mice receiving HA-PCL-DOX. The aforementioned lower liver accumulation could be ascribed to lower immunogenicity of hyaluronic acid shell. The most important feature of these systems was concerning drug residence time in liver which significantly decreased by 24 h for HAPCL-DOX formulation while in PEG-PCL-DOX treated mice, the DOX intensity in liver tissue increased with no detectable reduction. Thus, it could be suggested that hepatic processing and fast degradation of HA-PCL-DOX polymersomes was the main clearance mechanism of the hyaluronan-based vesicular system from the liver tissue up to 24 h.

23 | P a g e

A

B

****

150

****

Free DOX PEG-PCL-DOX HA-PCL-DOX

100

DOX intensity after 24H post-injection ****

150

**** Free DOX PEG-PCL-DOX HA-PCL-DOX

100

Tissue

ng Lu

n

ne y K id

rt

ee Sp l

ea

r H

Li ve

ey

ng Lu

rt

en

id n K

Sp le

H ea

or

ve r Li

Tu m

or

50

50

Tu m

DOX intensity

D

DOX intensity after 12H post-injection

DOX intensity

C

Tissue

Figure 8. Ex vivo fluorescence imaging of mice organs as well as tumor tissue. 12 h post-injection (A) and 24 h post-injection (B) of free DOX, HA-PCL-DOX and PEG-PCL-DOX. The intensity of DOX in mice organs and tumor tissue 12 h post-injection (C) and 24 h post-injection (D) of free DOX, HAPCL-DOX and PEG-PCL-DOX.

3.9.

In vivo antitumor efficacy

The potential of DOX-loaded HA-PCL nanoparticles to improve growth suppression of 4T1 tumor was assessed in ectopic model of 4T1 tumor-bearing BALB/c mice after single-dose intravenous administration of either HA-PCL-DOX, PEG-PCL-DOX or free DOX (DOX equivalent concentration: 5 mg/kg). Obtained results demonstrated a significant inhibition of tumor growth after administration of either HA-PCL-DOX or PEG-PCL-DOX when compared with free DOX and control group which was likely due to EPR. Moreover, the significant tumor inhibition effect of HA-PCL-DOX in comparison with PEG-PCL-DOX was attributed to targeting capability of HA-PC-DOX to CD44 receptor of 4T1 tumor cells which increased the accumulation of the formulation within the tumor (Fig. 9A). As a clinical sign of toxicity, the body weight of mice was monitored in all groups. According to the results, mice injected with either free DOX or PEG-PCL-DOX were demonstrated a body weight loss 3 days’ post-administration in comparison with mice in HA-PCL-DOX and 24 | P a g e

control groups which further confirmed the systemic toxicity of free DOX and PEG-PCLDOX. The systemic toxicity of PEG-PCL-DOX in term of body weight loss as compared with HA-PCL-DOX could be ascribed to higher release rate of DOX through this system which resulted in greater loss of body weight (Fig. 9B). The significant inhibition of tumor growth in HA-PCL-DOX-treated mice eventuated in statistically longer median survival time when compared with either PEG-PCL-DOX, free DOX or saline treated groups. The survival percentage of 4T1 tumor-bearing mice injected by either PEG-PCL-DOX, HA-PCL-DOX or free DOX with administration of a 5 mg/kg dose during a 35-day period after administration were presented in Figure 9C. In this regard, only one mouse remains alive with either 5 mg/kg free DOX or saline administration after 26 and 25 days, respectively. Three out of five mice receiving PEG-PCL-DOX stayed alive up to day 35. Although, all mice in the HA-PCL-DOX- treated group remained alive until day 35. The shorter time of survival for the free DOX-treated group when compared with the group injected by HA-PCL-DOX (statistically significant) could be ascribed to severe toxicity of free DOX. On the other hand, our observations did not exhibit any significant difference between median survival times in mice injected by either free DOX or saline.

25 | P a g e

A

Tumor Volume-Balb/C Tumor Volume-Balb/C Control Control

1200

1400

Free Dox

1000

1200

Free Dox HA-PCL-DOX HA-PCL-DOX PEG-PCL PEG-PCL

Tumor Volume (mm3)

Tumor Volume (mm3)

1400

800 600 400 200

1000 800 600 400 200

0

1

3

05

8 10 12 15 1 Time 3 (Day) 5 8

17 10

19 12

21 15

17

19

21

Time (Day)

Body weight

B

Control Free Dox HA-PCL-DOX

Body Weight (g)

24 22

PEG-PCL-DOX 20 18

h 3t

5t h 8t h 10 th 12 th 15 th 17 th 19 th 21 th

h 1t

16

Time (Days)

Survival Percent

C

Control Free DOX HA-PCL-DOX PEG-PCL-DOX

Survival Percent %

100

50

0

0

10

20

30

40

Time (Days)

Figure 9. Tumor growth patterns of ectopic model of 4T1 tumor in mice receiving intravenous injection of either HA-PCL-DOX, PEG-PCL-DOX or free DOX (equivalent DOX concentration: 5 mg/kg, n=5) (A); The body weight (g) of 4T1 tumoric mice during 21 days post single dose injection of either HAPCL-DOX, PEG-PCL-DOX or free DOX (equivalent DOX concentration: 5 mg/kg, n=5) (B); Survival percent of 4T1 tumoric mice during 35 days post single dose injection of either HA-PCL-DOX, PEGPCL-DOX or free DOX (equivalent DOX concentration: 5 mg/kg, n=5).

26 | P a g e

3.10.

Histopathological evaluation

To assess the ability of exerting necrosis in tumor tissue, 30 days after injection of either free DOX, HA-PCL-DOX, PEG-PCL-DOX or 0.9% NaCl solution as control group, the tumor tissue of each group was removed and H&E staining was performed. Figure 10 demonstrated major area of necrosis in tumors of mice receiving HA-PCL-DOX as compared with those treated with either free DOX or PEG-PCL-DOX.

Figure 10. Hematoxylin and eosin staining of tumor tissue of 4T1 turmoric mice 30 days’ postadministration of either 0.9% NaCl solution as control, free DOX, HA-PCL-DOX or PEG-PCL-DOX (equivalent DOX concentration: 5 mg/kg). Bright microscopy imaging was performed at 10× & 40× magnifications.

To evaluate the acute toxicity, the tissues of major organs were obtained from liver, spleen, heart, lung, kidneys after injection of either free DOX, HA-PCL-DOX, or PEG-PCL-DOX NPs. Thirteen days post intraveneous injection, oragns were removed. As shown in Figure 11, HA-PCL-DOX and PEG-PCL-DOX did not show any sign of toxicity in major organs. Moreover, we did not observe any change in their body weight and drinking/eating behavior.

27 | P a g e

Figure 11. Hematoxylin and eosin staining of major organs (kidney, heart, lung, spleen and liver) of 4T1 tumoric mice (30 days post- administration of either 0.9% NaCl solution as control, free DOX, HA-PCL-DOX or PEG-PCL-DOX (equivalent DOX concentration: 5 mg/kg).

Previously, heart damage in cancer patients receiving DOX was reported and documented (Rahman et al., 2007). Based on our observation, as represented in Figure 12, in contrast to heart tissue of mice receiving free DOX, no remarakable pathological alteration was detected in the heart tissues of mice injected by either PEG-PCL-DOX, HA-PCL-DOX or saline. Thus, both PEG-PCL-DOX and HA-PCL-DOX did not show any sign of cardiac toxicity in vivo in comparison with free DOX confirming the safty of these formulations.

28 | P a g e

Figure 12. Mice myocardium illustration of 4T1 turmoric mice 30 days’ post-administration of either 0.9% NaCl solution as control, free DOX, HA-PCL-DOX or PEG-PCL-DOX (equivalent DOX concentration: 5 mg/kg).

Until now, FDA-approved liposomal formulation of doxorubicin, Doxil has been extensively implemented in clinic, although it could not provide a desirable sustained-release characteristic of drug which represents as a limitation and disadvantage of Doxil (Alibolandi et al., 2017). Obtained results of the current investigation demonstrate that biodegradable sustained release HA-PCL nanopolymersomal formulation of DOX introduced ideal therapeutic index with selective DOX delivery to tumor tissue with no systemic toxicity after administration. 4. Conclusion Herein, we developed a hyaluronan-PCL polymersomal delivery system for targeted DOX transportation to CD44 positive murine 4T1 and human MCF-7 mammary cancer cell lines. The synthesized formulation could carry the drug with high encapsulation efficacy to the target site. It was accumulated in cancer cells at higher levels than in normal cells compared to free DOX and PEG-PCL-DOX formulation. Moreover, in vivo evaluations demonstrated that the HA-PCL-DOX had significant higher therapeutic index in comparison with PEG-PCL-DOX while illustrating the same safety profile. Thus, the developed hyaluronan-PCL polymersomes with targeting ability could be considered as an ideal candidate for DOX delivery in cancer treatment. Acknowledgments This work was funded by the Mashhad University of Medical Sciences, Mashhad, Iran (Grant number # 941379. This study was partly based on the PhD thesis of Dr. Mahsa Shahriari. Conflict of interest The authors declare that they have no confidence of interest.

29 | P a g e

References Alibolandi, M., Abnous, K., Hadizadeh, F., Taghdisi, S.M., Alabdollah, F., Mohammadi, M., Nassirli, H., Ramezani, M., 2016. Dextran-poly lactide-co-glycolide polymersomes decorated with folate-antennae for targeted delivery of docetaxel to breast adenocarcinima in vitro and in vivo. Journal of Controlled Release 241, 45-56. Alibolandi, M., Abnous, K., Mohammadi, M., Hadizadeh, F., Sadeghi, F., Taghavi, S., Jaafari, M.R., Ramezani, M., 2017. Extensive preclinical investigation of polymersomal formulation of doxorubicin versus Doxil-mimic formulation. Journal of Controlled Release 264, 228-236. Cazzaniga, M.E., Dionisio, M.R., Riva, F., 2017. Metronomic chemotherapy for advanced breast cancer patients. Cancer Letters 400, 252-258. Chen, C., Lu, L., Yan, S., Yi, H., Yao, H., Wu, D., He, G., Tao, X., Deng, X., 2018. Autophagy and doxorubicin resistance in cancer. Anti-cancer drugs 29, 1-9. Franco, Y.L., Vaidya, T.R., Ait-Oudhia, S., 2018. Anticancer and cardio-protective effects of liposomal doxorubicin in the treatment of breast cancer. Breast Cancer: Targets and Therapy 10, 131. Jahangirian, H., Kalantari, K., Izadiyan, Z., Rafiee-Moghaddam, R., Shameli, K., Webster, T.J., 2019. A review of small molecules and drug delivery applications using gold and iron nanoparticles. International journal of nanomedicine 14, 1633. Jang, E., Lim, E.-K., Choi, Y., Kim, E., Kim, H.-O., Kim, D.-J., Suh, J.-S., Huh, Y.-M., Haam, S., 2013. π-Hyaluronan nanocarriers for CD44-targeted and pH-boosted aromatic drug delivery. Journal of Materials Chemistry B 1, 5686-5693. Jeannot, V., Mazzaferro, S., Lavaud, J., Vanwonterghem, L., Henry, M., Arboleas, M., Vollaire, J., Josserand, V., Coll, J.-L., Lecommandoux, S., 2016. Targeting CD44 receptorpositive lung tumors using polysaccharide-based nanocarriers: Influence of nanoparticle size and administration route. Nanomedicine: Nanotechnology, Biology and Medicine 12, 921-932. Jiang, T., Chen, G., Shi, X., Guo, R., 2019. Hyaluronic Acid-Decorated Laponite® Nanocomposites for Targeted Anticancer Drug Delivery. Polymers 11, 137. Kalyane, D., Raval, N., Maheshwari, R., Tambe, V., Kalia, K., Tekade, R.K., 2019. Employment of enhanced permeability and retention effect (EPR): Nanoparticle-based precision tools for targeting of therapeutic and diagnostic agent in cancer. Materials Science and Engineering: C. Karousou, E., Misra, S., Ghatak, S., Dobra, K., Götte, M., Vigetti, D., Passi, A., Karamanos, N.K., Skandalis, S.S., 2017. Roles and targeting of the HAS/hyaluronan/CD44 molecular system in cancer. Matrix Biology 59, 3-22. Kumar, A., Gautam, B., Dubey, C., Tripathi, P.K., 2014. A review: role of doxorubicin in treatment of cancer. International Journal of Pharmaceutical Sciences and Research 5, 4105.

30 | P a g e

Lee, H., Park, H., Noh, G.J., Lee, E.S., 2018. pH-responsive hyaluronate-anchored extracellular vesicles to promote tumor-targeted drug delivery. Carbohydrate polymers 202, 323-333. Li, X., Tsibouklis, J., Weng, T., Zhang, B., Yin, G., Feng, G., Cui, Y., Savina, I.N., Mikhalovska, L.I., Sandeman, S.R., 2017. Nano carriers for drug transport across the blood– brain barrier. Journal of drug targeting 25, 17-28. Lim, E.-K., Kim, H.-O., Jang, E., Park, J., Lee, K., Suh, J.-S., Huh, Y.-M., Haam, S., 2011. Hyaluronan-modified magnetic nanoclusters for detection of CD44-overexpressing breast cancer by MR imaging. Biomaterials 32, 7941-7950. Liu, C.C., Chang, K.Y., Wang, Y.J., 2010. A novel biodegradable amphiphilic diblock copolymers based on poly (lactic acid) and hyaluronic acid as biomaterials for drug delivery. Journal of Polymer Research 17, 459-469. Meng, F., Zhong, Z., Feijen, J., 2009. Stimuli-responsive polymersomes for programmed drug delivery. Biomacromolecules 10, 197-209. Müller, L.K., Landfester, K., 2015. Natural liposomes and synthetic polymeric structures for biomedical applications. Biochemical and biophysical research communications 468, 411-418. Nagini, S., 2017. Breast Cancer: Current Molecular Therapeutic Targets and New Players. Anti-cancer agents in medicinal chemistry 17, 152-163. Ngan, Y.H., Gupta, M., 2016. A comparison between liposomal and nonliposomal formulations of doxorubicin in the treatment of cancer: An updated review. Archives of Pharmacy Practice 7, 1. Rahman, A.M., Yusuf, S.W., Ewer, M.S., 2007. Anthracycline-induced cardiotoxicity and the cardiac-sparing effect of liposomal formulation. International journal of nanomedicine 2, 567. Shafei, A., El-Bakly, W., Sobhy, A., Wagdy, O., Reda, A., Aboelenin, O., Marzouk, A., El Habak, K., Mostafa, R., Ali, M.A., 2017. A review on the efficacy and toxicity of different doxorubicin nanoparticles for targeted therapy in metastatic breast cancer. Biomedicine & Pharmacotherapy 95, 1209-1218. Tahmasbi Rad, A., Chen, C.-W., Aresh, W., Xia, Y., Lai, P.-S., Nieh, M.-P., 2019. Combinational Effects of Active Targeting, Shape, and Enhanced Permeability and Retention for Cancer Theranostic Nanocarriers. ACS applied materials & interfaces. Wu, C.-H., Lan, C.-H., Wu, K.-L., Wu, Y.M., Jane, W.-N., Hsiao, M., Wu, H.-C., 2018. Hepatocellular carcinoma-targeted nanoparticles for cancer therapy. International journal of oncology 52, 389-401. Zhang, Y., He, Y., Lu, L.L., Zhou, Z.Y., Wan, N.B., Li, G.P., He, X., Deng, H.W., 2019. miRNA‐192‐5p impacts the sensitivity of breast cancer cells to doxorubicin via targeting peptidylprolyl isomerase A. The Kaohsiung journal of medical sciences 35, 17-23. Zhang, Y., Zhao, Q., Shao, H., Zhang, S., Han, X., 2014. Synthesis and characterization of star-shaped block copolymer sPCL-b-PEG-GA. Advances in Materials Science and Engineering 2014. 31 | P a g e

Zhao, N., Leng, Q., Woodle, M.C., Mixson, A.J., 2019. Enhanced tumor uptake and activity of nanoplex-loaded doxorubicin. Biochemical and Biophysical Research Communications.

Highlight 

Doxorubicin was encapsulated in hyaluronan-polycaprolactone polymersomes.



CD44-receptor mediated endocytosis was confirmed in 4T1 and MCF-7 cell lines.



Excellent therapeutic efficiency of this formulation was verified in 4T1 tumor model.



Ideal biodistribution of this formulation was also confirmed in 4T1 tumor model.

32 | P a g e

33 | P a g e

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

34 | P a g e