Folate receptor-targeted multimodal polymersomes for delivery of quantum dots and doxorubicin to breast adenocarcinoma: In vitro and in vivo evaluation

Accepted Manuscript Title: Folate Receptor-Targeted Multimodal Polymersomes for Delivery of Quantum Dots and Doxorubicin to Breast Adenocarcinoma: in vitro and in vivo Evaluation Author: Mona Alibolandi Khalil Abnous Fatemeh Sadeghi Hossein Hosseinkhani Mohammad Ramezani Farzin Hadizadeh PII: DOI: Reference:

S0378-5173(16)30040-0 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.01.040 IJP 15506

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

16-11-2015 14-1-2016 14-1-2016

Please cite this article as: Alibolandi, Mona, Abnous, Khalil, Sadeghi, Fatemeh, Hosseinkhani, Hossein, Ramezani, Mohammad, Hadizadeh, Farzin, Folate ReceptorTargeted Multimodal Polymersomes for Delivery of Quantum Dots and Doxorubicin to Breast Adenocarcinoma: in vitro and in vivo Evaluation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.01.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.

Folate Receptor-Targeted Multimodal Polymersomes for Delivery of Quantum Dots

 1

and Doxorubicin to Breast Adenocarcinoma: in vitro and in vivo Evaluation

 2  3

Mona Alibolandi1, Khalil Abnous1, Fatemeh Sadeghi2, Hossein Hosseinkhani3,

 4

Mohammad Ramezani1,4*, Farzin Hadizadeh5*

 5  6

1

Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences,

 7

Mashhad, Iran

 8

2

 9

Targeted Drug Delivery Research Center, School of Pharmacy, Mashhad University of Medical

Sciences, Mashhad, Iran

  10

3

  11

Graduate Institute of Biomedical Engineering, National Taiwan University of Science and

Technology, Taipei, Taiwan

  12

4

  13

Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences,

Mashhad, Iran

  14

5

  15

Biotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences,

Mashhad, Iran

  16   17   18

*Corresponding authors:

  19

Prof. Farzin Hadizadeh, Biotechnology Research Center, School of Pharmacy, Mashhad   20 University of Medical Sciences, P.O. Box 9196773117, Mashhad, Iran. Fax: +98 51   21 37112470; Tel: +98 51 37112420, E-mail: [email protected]

  22

Prof. Mohammad Ramezani, Pharmaceutical Research Center, School of Pharmacy,   23 Mashhad University of Medical Sciences, Mashhad, Iran. Fax: +98 51 37112470; Tel: +98 51   24 37112471, E-mail: [email protected]

  25   26

   | P a g e 1  

  27

Graphical abstract

  28   29   30   31

Highlights

1. Hydrophobic DOX and hydrophilic MSA-capped QD were encapsulated in the   32 bilayer and core of the PEG-PLGA nanopolymersomes, respectively.

  33

2. To achieve active cancer targeting in vitro and in vivo, QD and DOX–encapsulated   34 nanopolymersomes were conjugated with folate for folate–binding protein receptor–   35 guided delivery.

  36

3. In vivo experiments illustrated a high potential of the prepared targeted theranostic   37 nanoplatform in the treatment and imaging of breast cancer.

  38  

  39   40   41   42

   | P a g e 2  

  43

Abstract

In this study, we report the design and delivery of tumor-targeted, quantum dot (QD) and   44 doxorubicin (DOX)-encapsulated PEG-PLGA nanopolymersomes (NPs) for the imaging and   45 chemotherapy of breast cancer. To achieve active cancer targeting, QD and DOX–   46 encapsulated NPs were conjugated with folate for folate–binding protein receptor–guided   47 delivery, which overexpressed in many cancer cells. Hydrophobic DOX and hydrophilic   48 MSA-capped QD were encapsulated in the bilayer and core of the PEG-PLGA   49 nanopolymersomes, respectively.

  50

The data show that the formulated NPs sustained DOX release for a period of 12 days.   51 Fluorescence microscopy and MTT assay demonstrated that the developed folate-targeted   52 DOX-QD NPs had higher cytotoxicity than non-targeted NPs and the free form of the drug;   53 moreover, they preferentially accumulated in 4T1 and MCF-7 cells in vitro.

  54

In vivo experiments including whole organ tissue–homogenate analysis and organ   55 fluorescence microscopy imaging of BALB/c mice bearing 4T1 breast adenocarcinoma   56 showed that the folate receptor–targeted QD encapsulated NPs accumulate at tumor sites 6 h   57 following intravenous injection. Acute toxicity studies of the prepared targeted QD-loaded   58 NPs showed no evidence of long-term harmful histopathological and physiological effects on   59 the treated animals. The in vivo tumor inhibitory effect of folic acid (FA)-QD-DOX NPs   60 demonstrated an augmented therapeutic efficacy of targeted formulation over the non-   61 targeted and free drug. The data obtained illustrate a high potential of the prepared targeted   62 theranostic nanoplatform in the treatment and imaging of breast cancer. This study may open   63 new directions for preparation of QD-based theranostic polymersomes for clinical   64 application.

  65

Keywords: Nanopolymersome, Doxorubicin, Quantum dot, Breast cancer, 4T1, PEG-PLGA

  66   67

   | P a g e 3  

  68

1. Introduction

Breast cancer is one of the most common malignancies in women worldwide [1]. Standard   69 treatment of breast carcinoma involves chemotherapy alone or aggressive surgery followed   70 by chemotherapy [2]. Metastases and relapses cannot be efficiently cured by surgery or   71 radiation and require both extensive diagnosis and chemotherapy.

  72

In recent years, there has been a growing focus on the application of theranostic systems and   73 accomplishment of effective diagnosis, along with anti-cancer therapies with fewer side   74 effects [3, 4]. The establishment of such theranostic systems helps in cancer chemotherapy,   75 the early detection of cancer, and the measurement of the therapeutic response during the   76 therapy period. In this regard, nanoparticulate systems have ideal properties to develop novel   77 nanoplatform formulations that could efficiently integrate diagnostic and therapeutic   78 implementation in oncology [5]. Moreover, the incorporation of imaging agents would   79 represent a real-time, noninvasive evaluation opportunities for tumor physiology, drug   80 delivery, and therapeutic response [6].

  81

The efforts to generate theranostic systems have resulted in the development of various   82 nanoparticulate

systems

comprising

micelles,

liposomes,

inorganic

nanostructures,   83

dendrimers, and nanocapsules [7-11]. Among these, polymeric vesicles (polymersomes)   84 serve as a versatile carrier that can encapsulate multiple imaging modalities along with a drug   85 payload [12].

  86

Polymersomes are self-assembled polymeric vesicle in the form of core/shell nanostructures   87 which was made from amphiphilic copolymers in aqueous environment [13-15].   88 Polyethylene glycol (PEG)-poly(D,L-lactic-co-glycolic acid) PLGA-, PEG-PLA-, and PEG-   89 PCL‐based polymersomes have demonstrated a great strength in delivering both hydrophobic   90 and hydrophilic drugs and have been investigated comprehensively owing to their attractive   91 characteristics, which fulfill the requirements of efficient drug delivery [16-19]. The   92

   | P a g e 4  

hydrophobic bilayer of the polymersomes has been used extensively for encapsulation of   93 various non-polar drugs like paclitaxel, amphotericin-B, and docetaxel, while the interior   94 compartment of polymersomes has been employed for the encapsulation of polar drugs [20-   95 22]. Polymersomes usually control the release of the encapsulated hydrophobic–hydrophilic   96 drugs in a sustained manner and enhance the therapeutic index. However, the hydrophilic   97 PEG chains on the surface of PEG-polyester-based polymersomes impart excellent colloidal   98 stability while preventing the binding of serum proteins (opsonization) on the polymersome   99 surface, thereby avoiding rapid clearance from the blood stream by macrophages [23]. In   100 other words, the presence of PEG on the nanoparticle surface and the nanoscale size of the   101 polymersomes result in a very efficient passive accumulation of these particulate systems in   102 the tumor via the enhanced permeability and retention (EPR) effect [24]. However, a better,   103 more selective targeting system is required to increase intracellular uptake of nanocarriers   104 and their cargo within cancerous cells in tumor tissues [25].

  105

Numerous ligands targeting tumor cell–specific receptors have been conjugated on the   106 surface of nanoparticles to carry them within cells via receptor-mediated endocytosis. Among   107 these, folate has been extensively evaluated as a targeting ligand for numerous nanoplatforms   108 and the obtained results have shown its potency as a targeting ligand for various carcinomas   109 [26, 27]. Folate-binding protein, a transmembrane glycoprotein, is a cell-surface receptor for   110 folate that has been demonstrated to be overexpressed in human tumors comprising ovarian   111 and breast cancers, while exhibiting a very low level of expression in normal healthy tissues   112 [28-30]. In this regard, folic acid (FA) has been covalently conjugated to pegylated   113 doxorubicin (DOX) or different drug-encapsulated nanoparticles, including micelles,   114 liposomes, and nanospheres, to selectively target tumor tissues [31].

  115

Here, we report an efficient theranostic folate–conjugated targeted nanopolymersome (NP)   116 system encapsulating the anti-cancer drug DOX (for therapy) and MSA-capped CdTe   117

   | P a g e 5  

quantum dots (QDs; for diagnosis). Fluorescent QDs have been implemented as optical   118 biological probes because of their high photostability and unique optical properties   119 comprising a wide absorption bandwidth and narrow symmetric emission bandwidth [32].

  120

Hydrophilic MSA-capped CdTe QDs can be easily encapsulated in the core of the   121 polymersomes because of their ultra-small dimension in order to reduce cytotoxicity and side   122 effects. DOX has been commonly used as a routine anti-cancer agent in combined   123 chemotherapy for patients with breast cancer since 1982 [33]. In our polymersome   124 formulation, we encapsulated DOX in the bilayer and QDs at the core of the polymersomes;   125 then, folate was conjugated on the surface of the 100% pegylated NPs in order to prepare an   126 active targeting theranostic formulation.

  127

Previously, similar approaches were used where QDs and drugs were encapsulated in   128 different kinds of nanoparticles [34, 35]. In this study, we exploited PEG-PLGA   129 polymersomes for the encapsulation of DOX and QDs in the bilayer and core, respectively, of   130 the folate-conjugated polymeric vesicle.

  131

The release kinetics of the prepared formulation was examined to verify the sustained release   132 of DOX from the prepared formulation. The therapeutic and diagnostic efficacies of the   133 prepared formulations were tested in in vitro using 4T1 and MCF-7 cell lines. The selectivity,   134 biodistribution, and kinetics of the prepared nanoprobes (QD-loaded NP and folate-   135 conjugated QD-loaded NP) were evaluated in a mouse 4T1 tumor model. At the final stage,   136 the chemotherapeutic potential of the prepared theranostic formulations was investigated in   137 the 4T1 mouse breast cancer tumor model.

  138   139

2. Experimental

  140

2.1.Materials PLGA

(average

Mw:

hydroxysulfosuccinimide

10,000 (NHS),

Da;

lactic

1-ethyl

acid:

glycolic

acid

=

3-(3-dimethylaminopropyl)

75:25),

N-   141

carbodiimide   142

   | P a g e 6  

hydrochloride

(EDC),

N,N'-dicyclohexylcarbodiimide

(DCC),

FA,

and

3-(4,5-   143

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma   144 Aldrich (Schnelldor, Germany). Heterofunctional PEG polymer with a terminal amine and   145 carboxylic acid functional groups (HCl*NH2-PEG-COOH, Mw: 3500) was purchased from   146 JenKem Technology USA (Beijing, China). Doxorubicin hydrochloride (DOX) was   147 purchased from Euroasia Co., Ltd. (Delhi, India), and hydrophobic DOX was prepared at our   148 laboratory by neutralizing DOX*HCl with triethylamine. Roswell Park Memorial Institute   149 (RPMI) 1640 medium, fetal bovine serum (FBS), penicillin–streptomycin, and trypsin were   150 purchased from GIBCO (Darmstadt, Germany). Other solvent and chemical reagents were   151 procured from Merck (Darmstadt, Germany) without further purification.

  152

2.2.Synthesis and characterization of PEG-PLGA copolymer [36]

  153

One gram of PLGA-COOH (average MW = 10 kDa), NHS and EDC were gently stirred in 4   154 mL of dichloromethane (1:8:8 PLGA:NHS:EDC molar ratio) at room temperature for 24 h to   155 prepare PLGA-NHS. To remove residual NHS and EDC, activated PLGA-NHS was   156 precipitated with cold diethyl ether and washed three times with cold freezing solution   157 containing 80% diethyl ether and 20% methanol.

  158

Vacuum-dried PLGA-NHS was dissolved in chloroform (5 mL) and then HCl*NH2-PEG-   159 COOH (1:1.2 PLGA:PEG molar ratio) and N,N-diisopropylethylamine (0.2 mmol) was   160 added to the polymer solution. After 24 h, the copolymer was precipitated with cold diethyl   161 ether and washed three times with methanol:diethyl ether solution (30:70). The final PLGA-   162 PEG block copolymer was freeze dried for 48 h and stored at -20°C until use.

  163

The 1H-NMR (proton nuclear magnetic resonance) spectra of the PEG-PLGA copolymer in   164 deuterated chloroform was recorded at room temperature using a Bruker Avance 300 MHz   165 NMR spectrometer (Rheinstetten, Germany) to verify PEG conjugation to PLGA. The 1H   166 NMR spectra were implemented to calculate the Mn of the copolymers from the integration   167

   | P a g e 7  

ratio of the resonances at 5.4 ppm and 4.6 ppm originating from the CH of the lactic acid   168 (LA) and CH2 of the glycolic acid (GA) units, respectively, and the integration ratio of the   169 resonance at 3.8 ppm originating from the CH2 of the ethylene glycol according to a   170 previously established method [37].

  171

2.3.Preparation of aminated folic acid (FA)

  172

FA (1 mmol) was dissolved in 10 mL dimethyl sulfoxide of (DMSO) and incubated with   173 DCC (1 mmol) and NHS (2 mmol) at room temperature for 20 h. Ethylene diamine (EDA)   174 and pyridine (200 µL) were added to the reaction mixture and further reacted at room   175 temperature overnight. The aminated FA was precipitated and washed repeatedly with excess   176 acetonitrile to remove residual DCC and NHS. The final product was freeze dried for 24 h   177 and stored at -20ºC until use.

  178

2.4.Synthesis and characterization of FA-conjugated PEG-PLGA (FA-PEG-PLGA)

  179

COOH-PEG-PLGA was dissolved in chloroform and activated with EDC and NHS (COOH-   180 PEG-PLGA:EDC:NHS 1:8:8 molar ratio). Activated NHS-PEG-PLGA was dissolved in   181 DMSO and aminated FA was slowly added to the polymer solution. The reaction mixture   182 was stirred at room temperature for 18 h.

  183

The solution was dialyzed (MWCO: 6,000–8,000 kDa) against distilled water for 48 h to   184 remove the unreacted aminated FA and freeze dried. The structure of PLGA-PEG-FA was   185 confirmed by 1H-NMR spectroscopy (VarianINOVA-400 MHz NMR spectrometer) in   186 DMSO-d6. The amount of FA conjugated to PLGA-PEG-FA was determined using an   187 ultraviolet (UV)-visible calibration curve of FA established in DMSO at 358 nm.

  188

The presence of amide linkages in PLGA-PEG-FA was investigated using a Paragon 1000   189 (Perkin Elmer, USA) Fourier transform infrared (FT-IR) spectrophotometer. Approximately   190 10 mg of PEG-PLGA or PLGA-PEG-FA was dissolved in chloroform and dropped on the   191

   | P a g e 8  

horizontal face of the NaCl crystal to prepare the polymer thin film. All reactions which   192 performed in the current study were illustrated in Figure 1.

  193

2.5.Quantum dot (QD) synthesis

  194

The CdTe QDs were prepared according to our previous study [38]. Briefly, the reaction took   195 place in borate–acetic acid buffer (pH=6, 100 mL) containing CdCl2 (20 mg, 1 mM),   196 Na2TeO3 (4.4 mg, 0.2 mM), and mercaptosuccinic acid (45 mg, 3 mM) under microwave   197 irradiation (400 W) at 100°C for 30 min.

  198

2.6.Nanoparticle preparation

  199

Blank PEG-PLGA NPs and QDs-loaded NPs (QD-NPs) were prepared using the double   200 emulsion (W/O/W) method. Briefly, 20 mg of PEG-PLGA or FA-PEG-PLGA dissolved in 1   201 mL of methylene chloride (70%):acetone (30%) and 0.4 mL of 1% w/v bovine serum   202 albumin solution or QD solution in 1% w/v bovine serum albumin (10 mg/mL) were   203 transferred to a centrifuge tube, and the mixture was emulsified by sonication for 3 min while   204 the temperature was kept at 20ºC. Then, the emulsion and 4 mL of 5% polyvinyl alcohol   205 (PVA) were emulsified by sonication for 5 min. The emulsion was then slowly dropped into   206 10 mL of 1% PVA and stirred for 6 h at room temperature. After vacuum evaporation of the   207 solvent, the NPs were collected by centrifugation at 15,000 rpm for 20 min at room   208 temperature and washed twice using distilled water. In the final stage, the size-exclusion   209 chromatography (Sephadex 50) column was used to further remove non-encapsulated QDs.

  210

QD- and DOX-loaded NPs (QD-DOX-NPs) were prepared using the same double emulsion   211 method. After the first emulsification, the mixture and 4 mL of 5% PVA were stirred for 3   212 min at room temperature. Meanwhile, 0.2 mL of DOX-dissolved methylene chloride (1 mg)   213 was added slowly and then emulsified again. The subsequent steps were identical to the   214 preparation of PEG-PLGA NPs and QD-NPs.

  215

   | P a g e 9  

Distinctively, DOX-loaded NPs (DOX-NPs) were produced using an emulsion/solvent   216 evaporation technique. Twenty milligrams of copolymer and 1 mg of DOX were dissolved in   217 1 mL of methylene chloride (70%):acetone (30%). The solution was stirred for 10 min at   218 room temperature and emulsified by sonication in 10 mL of aqueous solution with 5% PVA.   219 The emulsified solution was stirred for 6 h at room temperature. After vacuum evaporation of   220 the solvent, the NPs were collected by centrifugation at 15,000 rpm for 20 min at room   221 temperature and washed twice using distilled water.

  222

2.7.Determination of folate content on the surface of nanoparticles

  223

The amount of folate on the surface of blank NPs prepared by double emulsion (W/O/W) and   224 emulsion/solvent evaporation methods was determined via UV spectroscopy. Measurements   225 were performed in DMSO solvent. The nanoparticle solution in DMSO was analyzed by   226 evaluating the absorbance at 358 nm (FA ε = 15760 M-1 cm-1).

  227

2.8.Measurements of particle size distribution and zeta potential

  228

The particles’ size polydispersity index and surface charge (zeta potential, mV) were   229 determined using a ZetaSizer (NANO-ZS, Malvern, UK). Briefly, the particle suspensions in   230 deionized water (1 mg/mL) were analyzed using a ZetaSizer equipped with a 4-mW He-Ne   231 laser operated at 633 nm through back-scattering detection at a scattering angle of 90°. All of   232 the measurements were performed in triplicate at 25±3°C.

  233

2.9.Evaluation of encapsulation efficiency of QDs and DOX

  234

The amount of DOX and/or QDs entrapped in either single or co-encapsulated NPs was   235 estimated by fluorescence spectrophotometer (Synergy H4 Hybrid Multi-Mode Microplate   236 Reader, Biotek, Model: H4MLFPTAD). Briefly, ∼1 mg of lyophilized DOX- and/or QD-   237 loaded NPs was dissolved in 1 mL of DMSO. The solution was sonicated for 5 min in an ice   238 bath. It was used to estimate the encapsulated molecules in NPs by fluorescence   239

   | P a g e 10  

spectrophotometry at λex = 485 nm and λem = 591 nm for DOX and at λex = 360 nm and λem =   240 535 for QDs.

  241

2.10.

  242

Morphological characterization of the prepared formulations

Transmission electron microscopy (TEM) was performed to investigate the size and   243 homogeneity of the prepared free QD and QD- or QD-DOX-loaded nanopolymersomes using   244 a high-resolution electron microscope (HR-TEM; JEOL-2100) operated at 200 kV with a   245 Gatan Orius SC600 CCD camera. The samples for TEM observation were prepared as   246 follows: The suspension of NPs (0.5 mg/mL) or QDs (1 mg/mL) was dropped onto copper   247 grids coated with an amorphous carbon film and dried thoroughly in an electronic drying   248 cabinet at a temperature of 25°C and a relative humidity of 45%.

  249

2.11.

  250

Optical characterization of QD-loaded NPs

The optical analysis for QDs and their encapsulated form were performed using a Jasco FP-   251 6200 spectrofluorometer (Tokyo, Japan), using an excitation wavelength of 360 nm.

  252

2.12.

  253

In vitro drug release

For the in vitro DOX release study, drug-loaded nanoparticles (30 mg) were reconstituted in   254 phosphate-buffered saline (PBS; 5 mL, pH 5.5 or 7.4) and transferred to dialysis bags   255 (MWCO: 3,500 Da) placed in 50 mL of PBS containing 0.1% Tween 80 with stirring at 80   256 rpm/37ºC. At appropriate intervals, 1 mL of release medium was withdrawn and replaced by   257 the addition of 1 mL of fresh medium. The samples were analyzed in a 96-well black bottom   258 plate using a Synergy H4 Hybrid Multi-Mode Microplate Reader (Biotek, Model:   259 H4MLFPTAD) with λexcitation and λemission values set to 485 and 591 nm, respectively. Then,   260 the accumulative ratios of the released DOX were calculated as a function of time. All of the   261 assays were performed in triplicate.

  262   263   264

   | P a g e 11  

2.13.

  265

Cellular uptake

The cellular uptake of targeted or non-targeted QD- and DOX-loaded NPs by MCF-7 and   266 4T1 was evaluated using an Olympus IX81 inverted fluorescence microscope equipped with a   267 DP26 Olympus camera (Tokyo, Japan). The collagen-treated cover slips (0.1% collagen in   268 acetic acid) were deposited in the wells of a 6-well plate. MCF-7 and 4T1 cells were seeded   269 in 6-well plates at 1 × 105 cells per well and cultured for 24 h. The next day, the cells were   270 incubated for 2 h with FA-QD-DOX-NPs (targeted formulation), QD-DOX-NPs (non-   271 targeted formulation), and free QD+DOX (DOX concentration: 20 µg/mL, QD concentration   272 50 µg/mL). Then, the culture medium was removed, and the cover slips were washed five   273 times with cold PBS and fixed with 4% formaldehyde for 15 min. The cover slips were then   274 placed on a slide to analyze by fluorescence microscopy.

  275

To confirm the findings of the selective targeting of folate receptors on the MCF-7 and 4T1   276 cells by folate-targeted QD-DOX-loaded NPs, a competitive experiment was carried out.   277 Here, excessive amounts (1 mM) of free folic acid were added to each well 30 min before the   278 addition of the targeted formulation.

  279

2.14.

  280

Cytotoxicity of MSA-capped CdTe QDs

NIH-3T3 cells were cultured in the DMEM medium, supplemented with 10% (v/v) heat-   281 inactivated fetal bovine serum (FBS), 1% penicillin–streptomycin at 37°C, and 5% CO2.   282 NIH-3T3, 4T1 and MCF7 cells were seeded onto 96-well plates at 5×103 and incubated for 24   283 h. After 24 h of treatment with MSA-capped CdTe QDs (concentrations, 0.01–100 µg/mL),   284 cadmium, and telluride (concentrations, 0.01–100 µg/mL), the media were removed, washed   285 with PBS, replaced with fresh complete medium, and 20 µL of MTT (5 mg/mL in PBS)   286 solution was added to each well and incubated for a further 4 h in a humidified incubator. The   287 MTT solution was then aspirated from the wells using a vacuum, and 100 µL of DMSO was   288 added to each well. The absorbance at 570 nm with a reference wavelength of 630 nm was   289

   | P a g e 12  

measured using an Infinite® 200 PRO multimode microplate reader (Tecan Group Ltd.,   290 Männedorf, Switzerland).

  291

2.15.

  292

Cell viability assays of the prepared formulations

MCF-7 and 4T1 cells were cultured in the RPMI medium, supplemented with 10% (v/v)   293 heat-inactivated FBS, 1% penicillin–streptomycin at 37 °C, and 5% CO2. MCF-7 and 4T1   294 cells were seeded onto 96-well plates at 5×103 and incubated for 24 h. After 4 h of treatment   295 with targeted and non-targeted formulations (FA-QD-DOX-NPs, QD-DOX-NPs, FA-QD-NP,   296 QD-NP) and free DOX/free QD, the media were removed, washed with PBS, replaced with   297 fresh complete medium, and further incubated for 48 h at 37°C in a humidified incubator.   298 After incubation for 48 h, 20 µL of MTT (5 mg/mL in PBS) solution was added to each well   299 and incubated for a further 4 h. The MTT solution was then aspirated from the wells using a   300 vacuum and 100 µL of DMSO was added to each well. The absorbance at 570 nm with a   301 reference wavelength of 630 nm was measured using an Infinite® 200 PRO multimode   302 microplate reader (Tecan Group Ltd.).

  303

To confirm the findings of the selective targeting of folate receptors on the MCF-7 and 4T1   304 cells by FA-conjugated NPs; a competitive experiment was carried out. Here, excessive   305 amounts (1 mM) of free folic acid were added to each well 30 min before the addition of the   306 targeted formulation.

  307

2.16.

  308

Investigation of nanoprobe efficacy in vivo

All animal experiments were conducted with the approval of the Institutional Ethical   309 Committee and Research Advisory Committee of the Mashhad University of Medical   310 Sciences. For all chemotherapy experiments, BALB/c female mice (18–20 g) bearing 4T1   311 tumors were employed.

  312

A tumor cell suspension (2 × 105 cells per mouse) was prepared in a serum-free RPMI 1640   313 medium and was subcutaneously inoculated into the right flank of the mice. Two weeks after   314

   | P a g e 13  

inoculation, mice with 200 mm3 tumors (5 mice in each group) received 0.2 mL of free QD   315 or targeted and non-targeted QD-loaded nanopolymersomes (QD concentration, 18 mg/kg)   316 via a single tail vein injection. The injection of NaCl 0.9% solution was used as a negative   317 control.

  318

2.16.1. Tissue collection for quantitative fluorescence spectroscopy and qualitative   319   320

fluorescence microscopy imaging

Animals were euthanized after the injection of QD (n=3 at 6 h), QD-loaded NP (n=3 at 6 h)   321 and folate-conjugated QD–loaded NP (n=3 at 6 h) nanoprobes by overdosing them with ether   322 inhalation anesthesia. After euthanasia, the organs were harvested and washed three times   323 with PBS.

  324

The tissues were cut into two pieces. One piece of tissue was weighed and the tissue   325 homogenate was prepared using a tissue homogenizer with 1 M of 20 mM KH2PO4. After   326 three cycles of freezing in liquid nitrogen and thawing at 37°C, the cell lysate was   327 centrifuged for 10 min at 4,000 rpm at 4°C. The homogenate was transferred to a black flat-   328 bottomed 96-well plate and measured using a Synergy H4 Hybrid Multi-Mode Microplate   329 Reader (Biotek, model: H4MLFPTAD) with λex and λem values set to 360 and 535 nm,   330 respectively. All of the assays were performed in triplicate.

  331

The QD signal in each organ was estimated based on the initial weight of each organ and   332 fluorescent background of each organ in the control group. Another piece of tissue was   333 frozen in liquid nitrogen and stored at –80ºC until sectioned into 5–7 µm thick slices for   334 fluorescence microscopy.

  335

2.16.2. Evaluation of in vivo acute toxicity of free QDs and NP QD formulations

  336

Sixteen days after 10 consecutive intravenous injections of targeted and non-targeted   337 formulations and free QDs (equivalent QD concentration, 18 mg/kg, 6-day interval between   338 injections), BALB/c mice (5 mice in each group) were sacrificed via an overdose of ether   339

   | P a g e 14  

inhalation. The lungs, heart, liver, spleen, and kidneys were fixed in a 10% neutral-buffered   340 formalin solution and then embedded in paraffin. Embedded organs in paraffin were cut at 5   341 µm thick and stained with hematoxylin and eosin (H&E). The images were prepared at 10×   342 magnification. In addition, the toxicities of free QDs and folate receptor–targeted and non-   343 targeted QD-loaded NPs were evaluated by monitoring the body weight during 60 days. 2.17.

  344

The tumor inhibitory efficacy of folate receptor–targeted and non-targeted QD-   345   346

DOX–loaded PEG-PLGA NPs in a 4T1 murine breast carcinoma model

Mice with 80–100 mm3 subcutaneous 4T1 tumor (five mice in each group) received 0.2 mL   347 of free QD, free DOX, or folate receptor–targeted and non-targeted QD-DOX-loaded NPs   348 (equivalent DOX concentration, 7 mg/kg; equivalent QD concentration, 18 mg/kg) via a   349 single tail vein injection. Injection of NaCl 0.9% solution was used as negative control.

  350

The tumor volume was calculated using the a×b×w/2 formula where “a” is the largest   351 diameter, “b” is the smallest diameter, and “w” is the height of the tumor. In addition, the   352 toxicities of free DOX, free QDs, and QD-DOX-loaded NPs were evaluated by monitoring   353 the body weight and survival rates. In each experiment, the mice were followed up to 60 days   354 post-tumor induction or until one of the following conditions for euthanasia was met: (1) the   355 body weight of mice dropped below 20% of their initial weight; (2) their tumor diameter was   356 larger than 2 cm; (3) they became sick and unable to feed; or (4) they were found dead [36].

  357

2.17.1. Cardiac toxicity

  358

An autopsy was performed 30 days after a single-dose injection of targeted and non-targeted   359 formulations (FA-DOX-QD-NP, DOX-QD-NP) and frees DOX (equivalent DOX   360 concentration, 7 mg/kg; equivalent QD concentration, 18 mg/kg) to 4T1 tumor-bearing   361 BALB/c mice. Heart samples were collected for microscopic examination. Histological   362 analysis was performed as described in Section 2.16.2.

  363   364

   | P a g e 15  

2.18.

  365

Statistical analysis

A one-way analysis of variance (ANOVA) was used to analyze the present study’s data. A

  366

probability value of less than 0.05 was considered significant. Results are expressed as mean   367 ± standard deviation (SD) unless otherwise noted.

  368

3. Results and Discussion

  369

3.1.Synthesis and characterization of PEG-PLGA copolymer

  370

The PLGA-COOH and NH2-PEG-COOH polymers were used to synthesize PLGA-b-PEG   371 copolymer with terminal carboxylic acid groups (PLGA-PEG-COOH). PLGA-COOH was   372 preactivated to its succinimide using EDC and N-hydroxysulfosuccinimide (sulfoNHS) and   373 then

reacted

with

NH2-PEG3500-COOH.

The

resulting

PLGA-PEG-COOH

was   374

characterized by 1H-NMR (400 MHz, CDCl3),  5.18 (m, ((OCH(CH3)C(O)OCH2C(O))n-   375 (CH2CH2O)m),

4.8

(m,

((OCH(CH3)C(O)OCH2C(O))n-(CH2CH2O)m),

3.6

(s,   376

((OCH(CH3)C(O)OCH2C(O))n-(CH2CH2O)m), and 1.6 (d, ((OCH(CH3)C(O)OCH2C(O))n-   377 (CH2CH2O)m). By integrating the relative molecular weights and peaks, the conjugation   378 efficiency of COOH-PEG-NH2 to PLGA-COOH was estimated to be approximately 83%.

  379

3.2.Synthesis and characterization of FA-PEG-PLGA copolymer

  380

Two-step EDC/NHS chemistry allows the conjugation of amino groups of aminated folate to   381 the carboxyl groups of PEG-PLGA through an amide bond formation [36]. Conjugation of   382 folate to PEG-PLGA copolymer was verified by FT-IR analysis, and the very small amide   383 bond peaks at 1,626 cm (carbonyl, C=O) and 1,570 (amine C-N) in folate-conjugated PEG-   384 PLGA copolymer, which are absent in PEG-PLGA copolymer, confirmed the formation of   385 amide linkage following the conjugation reaction (Figure 2A, B).

  386   387   388

   | P a g e 16  

The reaction yield was calculated and found to be 80.23 µmol of folate per gram of   389 copolymer. 1H-NMR in DMSO-d6 was also carried out to verify the structure and purity of   390 the prepared FA-conjugated PEG-PLGA. The signals at 1–2 ppm and 4–5.5 ppm were due to   391 methyl and methylenic protons of the PLGA block, while signals at 3–4 ppm were due to the   392 methylenic protons of the PEG moiety. The characteristic peaks of FA aromatic protons were   393 observed at 6–9 ppm, which confirms the FA conjugation in the polymer (Figure 3).

  394

3.3.Preparation and characterization of DOX- and QD-loaded PEG-PLGA and FA-PEG-   395   396

PLGA NPs

The combination of diagnostic and therapeutic capabilities in a nanoplatform has attracted   397 increasing attention as a promising strategy to overcome the undesirable toxicity of   398 anticancer drugs. It also enables real-time follow-up of the patient response to therapy.   399 Therefore, it is crucial to explore a co-delivery system that could encapsulate an imaging   400 agent along with anticancer drugs in the same vehicle and carry them to the cancer tissue   401 simultaneously. In this regard, the co-encapsulation of molecules with different water   402 solubility characteristics is a key challenge in the development of co-delivery systems for   403 clinical applications [40, 41].

  404

The double emulsion method allows the formation of NPs with the ability to encapsulate both   405 polar and non-polar cargos [42]. In the current study, we used the double emulsion method to   406 encapsulate MSA-capped QDs (polar) and DOX (non-polar) with different water solubility   407 properties in PEG-PLGA polymersomes. Like other amphiphilic copolymers, PEG-PLGA   408 could be emulsified in an aqueous solution to form self-assembled structures [43].

  409

Previously, we proved that the PEG-PLGA block copolymer with a molecular weight of   410 PEG3500–PLGA10000 formed polymersomes in aqueous solutions [36]. In this regard, many   411 reports have demonstrated that in linear amphiphilic copolymers, a hydrophilic volume   412 fraction (fEO) of 25–40% results in self-assembled polymersomes [44]. Polymersomes share   413

   | P a g e 17  

many similarities with liposomes; however, they have several advantages over liposomes,   414 such as high stability and low permeability [45, 46]. It is also feasible that they can be   415 conjugated with different ligands for targeting delivery and that they can provide sustained   416 and controlled release of encapsulated polar and non-polar drugs in vivo [47-49].

  417

Prepared polymersomes should be <200 nm in size to evade detection and uptake by the   418 reticuloendothelial system (RES) and increase the circulation half-life [50]. Our resulting   419 polymersomes were characterized using a ZetaSizer apparatus. Table 1 summarizes the   420 average particle sizes of blank and formulated polymersomes. Blank PEG-PLGA and FA-   421 PEG-PLGA polymersomes showed an average size of 156.21 ± 0.21 nm and 152.5 ± 5.23   422 with polydispersity indexes of 0.13 and 0.121, respectively; the zeta potential was -23 mV for   423 both blank and formulated polymersomes. Folate present on the surface of blank   424 polymersome was quantitatively estimated by UV spectroscopy. The amount of folate on the   425 surface of nanoparticles was found to be 4.35 µmol of folate/gram of nanoparticles.

  426

The difference between the folate amount on the surface of blank nanoparticles prepared via   427 W/O/W or single emulsion method was not statistically significant.

  428

The size of QD-NPs was the same as that of blank NPs, indicating that the presence of QD in   429 the hydrophilic core of the polymersomes did not increase the volume. The obtained results   430 demonstrated that the encapsulation of DOX or co-encapsulation of DOX and QDs within   431 polymersomes slightly increased the size of nanoparticles with an appropriate polydispersity,   432 which might be attributed to the insertion of a hydrophobic drug (DOX) in the polymersome   433 bilayer, making the amphiphilic copolymers form larger structures.

  434

The morphology of QD-DOX-NPs and FA-QD-DOX-NPs, homogeneity, and bilayer   435 configuration were also investigated by high resolution TEM (Figure 4A, B). It was   436 confirmed that QD-DOX-NPs and FA-QD-DOX-NPs in the form of vesicles were dispersed   437 as spherical individual particles homogeneously distributed at a size of around 140–170 nm.

   | P a g e 18  

  438

Surface modification of PEG-PLGA NPs with folate had no significant effect on their   439 physical characteristics in terms of size, zeta potential, or morphology. A similar result was   440 obtained by Jiang et al. when they conjugated folate on the surface of nanoparticles [51].

  441

An ideal NP drug delivery system should encompass high encapsulation efficiency and high   442 loading content to decrease the vehicle quantity during drug-loaded NP administration [52].   443 In the current study, DOX and QDs were efficiently loaded in PEG-PLGA NPs, reaching a   444 loading of 40 µg of DOX and 100 µg of QD per milligram of NPs with encapsulation   445 efficiencies of 83–86% and 51–54%, respectively, at a copolymer:DOX:QD ratio of 20:1:4   446 (Table 1).

  447

Folate-targeted polymersomes exhibited similar drug entrapment efficiency to that of non-   448 targeted polymersome. In agreement with the obtained results, Misra et al. verified the   449 comparable loading efficiency of DOX in nuclear localization signal (NLS)-conjugated and   450 unconjugated NPs [53].

  451

3.4.Optical and structural characterization of QD-loaded PEG-PLGA polymersomes

  452

Figure 4A illustrates the emission spectra of free QDs, blank PEG-PLGA polymersome, and   453 QD-encapsulated polymersome at the excitation wavelength of 360 nm.

  454

The emission of QDs without any red shift at 535 nm in the QD-encapsulated polymersome   455 and the absence of this emission peak in the blank PEG-PLGA polymersome indicated the   456 loading of QDs in the aqueous interior compartment of polymersome without any changes in   457 the fluorescent characteristics of QDs. Based on the obtained results, QDs can be   458 encapsulated into PEG3500–PLGA10000 polymersomes without aggregation and alteration of   459 fluorescence properties of QDs.

  460

Figure 5A, B also demonstrates that the fluorescence spectra emitted from QDs encapsulated   461 polymersome are weaker than those from free QDs. This phenomenon is ascribed to the fact   462 that the QDs are encapsulated inside polymersome, which absorbs part of the excitation light.

   | P a g e 19  

  463

Figure 5C and D represent HR-TEM images of MSA-capped CdTe QDs before and after   464 polymersome encapsulation. The average diameter of the synthesized monodispersed QDs is   465 3 nm in size. Since the size of the MSA-capped CdTe QDs is relatively small, several QDs   466 were found to be encapsulated within a polymersome. As demonstrated in the TEM image,   467 several QDs are encapsulated in the interior compartment of polymersome vehicle and the   468 crystal nanostructure of individual QDs remains unaltered.

  469

3.5.In vitro drug release

  470

Sustained release of encapsulated drug from polymeric nanoparticles is a crucial factor for   471 the preparation of ideal chemotherapeutic formulations, as it facilitates delivery of a   472 continuous, constant amount of drug to the tumor. In this regard, it has been reported that   473 continuous low-dose administration of DOX has a better ability to induce apoptosis than a   474 single high-dose exposure of DOX [54]. Therefore, sustained release of DOX therapeutic   475 concentration in clinical chemotherapy would be desirable.

  476

As represented in Figure 6, the release profile of DOX from DOX-loaded NPs or DOX- and   477 QD-loaded NPs demonstrated a biphasic drug release profile comprising an initial burst   478 release followed by a slower continuous release phase over 12 days, indicating the typical   479 sustained and prolonged drug-release characteristics that depend on drug diffusion and matrix   480 erosion mechanisms. The initial burst drug release that was observed within 24 h in all   481 prepared formulations is a routine phenomenon among polymer-based matrixes [55].

  482

The burst release is related to the diffusion of the DOX adsorbed on the NP surface and/or to   483 the release of the encapsulated drug residing on the NP surface [36].

  484

The obtained results demonstrated that the co-encapsulation of DOX and QD did not exhibit   485 any influence on the release profile of the DOX from polymersome vehicle [56]. In contrast,   486 the release profile of DOX from folate-conjugated NPs was identical to that of unconjugated   487 nanopolymersome (data not shown).

  488

   | P a g e 20  

3.6.Cellular uptake of different QD-DOX-loaded nanoformulations by fluorescent   489   490

microscopy

Using the intrinsic fluorescence characteristics of DOX and QDs, a qualified analysis of the   491 cellular uptake pattern of FA-QD-DOX-NPs, QD-DOX-NPs, and free DOX/QDs was carried   492 out in the folate receptor overexpressing 4T1 and MCF-7 cells using a fluorescence   493 microscope. The relative cellular uptake of free DOX/QDs, QD-DOX-NPs, and FA-QD-   494 DOX-NPs is demonstrated in Figure 7 in terms of the fluorescence intensity displayed by the   495 cells. As shown in the figure, cellular uptake of FA-QD-DOX-NPs was higher than that of   496 unconjugated QD-DOX-NPs, as well as frees DOX/QDs, in both the 4T1 and MCF-7 cell   497 lines. However, there was no significant uptake of FA-QD-DOX-NPs compared to QD-   498 DOX-NPs in cells treated with free FA prior to the addition of FA-QD-DOX-NPs, as shown   499 by the fluorescence intensity. The above results clearly indicate that the superior uptake of   500 FA-QD-DOX-NPs in comparison to QD-DOX-NPs in the 4T1 and MCF-7 cell lines is due to   501 folate receptor–mediated endocytosis.

  502

The lower cellular uptake of QD-DOX-NPs in comparison with that of free DOX/QDs was   503 mainly due to the stability of the NP bilayer; this system’s controlled-release characteristics   504 and 100% pegylated surface of the nanoparticles decreased its in vitro cellular uptake.

  505

Numerous studies have evaluated the folate receptor–mediated uptake of drugs. In this   506 regard, Wang et al. verified the augmented uptake of folate-conjugated paclitaxel–loaded   507 micelles in comparison with unconjugated micelles in MCF-7 cells. They demonstrated that   508 implementing folate receptor–targeted carriers enhances drug uptake by the cancer cells that   509 overexpress folate receptor protein on their surfaces [57]. Moreover, the enhanced cellular   510 uptake level of folate-conjugated polymersomes may be ascribed to the lower exocytosis of   511 folate-conjugated polymersome in relation to unconjugated polymersome [58].

  512   513

   | P a g e 21  

  514

3.7.Cellular toxicity (MTT) of MSA-capped CdTe

Before in vivo application of the QD formulation, in vitro cytotoxicity evaluation of MSA-   515 capped QDs was precisely carried out on NIH-3T3, 4T1 and MCF-7 cells using MTT assay.   516 Previously, it was demonstrated that elemental cadmium and tellurium are cytotoxic to live   517 cells. Elemental cadmium was found to be toxic to NIH-3T3, 4T1 and MCF7 cells at a   518 concentration of 3, 1.25 and 2.5 µg/ml respectively. Elemental tellurium was found to be   519 cytotoxic at a concentration of 20, 15 and 18 µg/ml in NIH-3T3, 4T1 and MCF7 cells   520 respectively. However, MSA-capped CdTe QDs were nontoxic to NIH-3T3, 4T1 and MCF7   521 cells at a concentration of 50-60 µg/ml.

  522

Earlier, it was demonstrated that mercaptoacetic acid (MAA)-capped CdTe QDs were more   523 cytotoxic than MSA-capped CdTe QDs under similar treatment conditions [56]. Altogether   524 previous findings in this regard suggest that the surfactant types are pivotal role in elucidating   525 the toxicity level of the functionalized CdTe QDs [60, 61]. The obtained results demonstrated   526 that the all cells maintained 70%–80% viability even after being treated with 80-100 μg/mL   527 MSA-capped CdTe QDs for 24 h.

  528

3.8.Cell viability assays for the prepared formulations

  529

We further investigated in vitro cytotoxicity experiments on 4T1 and MCF-7 cell lines to   530 verify the biocompatibility of the QDs polymersomes and evaluate the therapeutic efficacy of   531 the encapsulated DOX. Figures 8 and 9 shows the results obtained from the cell viability   532 assay (MTT assay) carried out with the 4T1 and MCF-7 cell lines incubated with FA-QD-   533 DOX-NPs, QD-DOX-NPs, QD-NPs, FA-QD-NPs, and free DOX or free QDs. The   534 concentration of all of the samples was calculated based on the concentration of DOX present   535 in the polymersome formulation. The results clearly demonstrated the potential of this   536 theranostic polymeric vesicle.

  537

   | P a g e 22  

We also tested the cytotoxicity of targeted and non-targeted QD nanoformulations. QD-NPs   538 and FA-QD-NPs exhibited low cytotoxic effect, and the viability of cell lines was maintained   539 above 80% with treatment concentrations as high as 70 μg/mL (Figure 9A, B). This result   540 demonstrated the low toxicity of the QD-loaded polymersome formulation and suggested that   541 the cytotoxicity of the prepared theranostic system (QD-DOX-NPs) was mostly caused by the   542 encapsulated DOX.

  543

QD-DOX-NPs at a DOX concentration of > 35 µg/ml and 30 µg/ml showed approximately   544 50% cell survival in 4T1 (Figure 8A) and MCF-7 (Figure 8B), respectively, whereas at the   545 same concentration, free DOX showed just 34% and 26% cell survival in 4T1 and MCF-7   546 cell lines. The lower cellular uptake and cytotoxicity of QD-DOX-NPs in comparison with   547 free DOX is largely due to the stability of the NP bilayer and this system’s controlled-release   548 characteristics.

  549

FA-QD-DOX-NPs at a DOX concentration of 2.5 µg/ml (for the 4T1 cell line) and 1.2 µg/ml   550 (for the MCF-7 cell line) showed strong cytotoxicity, since only 50% cell survival was   551 evident. FA conjugation increased the cytotoxicity of nanoparticles into folate receptor–   552 overexpressing cell lines (MCF-7 and 4T1). Moreover, the FA-QD-DOX-NPs enhanced the   553 cytotoxicity to the MCF-7 and 4T1 tumor cells, while showing no efficacy towards the free   554 FA (1 mM)-treated cells. This is consistent with the results shown in Fig. 6, in which the FA   555 conjugation increased the uptake of FA-QD-DOX-NPs into MCF-7 and 4T1 cells but did not   556 have the same effect in cells treated with free FA. The results suggest that FA may selectively   557 enhance the delivery of anticancer drug to MCF-7 and 4T1 cancer cells.

  558

Based on the obtained results, we think that the prepared unique QD-based theranostic   559 polymersome formulation co-encapsulating MSA-capped QDs and DOX can be implemented   560 as a capable nanoplatform for simultaneous breast cancer imaging and therapy.

  561   562

   | P a g e 23  

  563

3.9.Nanoprobe delivery in vivo

In vivo organ distribution of targeted and non-targeted QD-NPs, FA-QD-NPs, and free QD   564 was carried out in a BALB/c mouse model of 4T1 tumor allografts. Mouse breast cancer 4T1   565 cells were implanted subcutaneously above the flank of the mice and allowed to grow. QD-   566 NPs, FA-QD-NPs, and free QDs were injected intravenously.

  567

The organs’ homogenate fluorescent quantity 6 h after injection showed a higher   568 accumulation of the FA-QD-NPs in the tumor and lower accumulation in other studied   569 organs when compared with the QD-NPs and frees QDs (Figure 10 A, B). The higher FA-   570 QD-NP fluorescence when compared to QD-NP fluorescence in the tumor tissues at 6 h   571 demonstrated the specific binding of FA-QD-NPs nanoprobes to the overexpressed FA   572 receptor in tumor tissues. In contrast, the fluorescence from QDs and QD-NPs in the tumor   573 tissues at 6 h post-injection time exhibited significant differences, indicating the rapid   574 clearance of free QD nanoprobes from tumor tissue.

  575

It was shown previously that the existence of a highly fenestrated blood vasculature in the   576 tumor tissues promotes the EPR effect and enhances uptake of nanoscale vehicles (less than   577 200 nm in size) into the tumor. Moreover, the poor lymphatic flow in the tumor tissue   578 increases the EPR effect and enhances retention of nanoparticles within the tumor tissue. In   579 contrast, vehicles less than 50 nm in size extravasate from the tumor tissue through the   580 fenestrated vessels of the tumor blood vascular system, and consequently, their retention time   581 at the tumor site decreases [62, 63].

  582

The biodistribution of QDs in the heart, lungs, kidney, and spleen did not show different   583 patterns in either the QD-loaded polymersome or folate conjugated QD-loaded NP groups   584 after 6 h. However, the uptake of QD by the kidney and liver was observed to be significantly   585 higher for free QDs compared to targeted and untargeted QD-loaded polymersomes at 6 h   586

   | P a g e 24  

after intravenous injection. Moreover, the folate-conjugated QD-loaded NP–treated group   587 exhibited higher accumulation in the liver compared to QD-loaded NPs.

  588

The uptake by the reticular endothelial system (RES), comprising the liver, at 6 h post-   589 injection was detected to be higher for the free QDs and folate-conjugated QD-loaded NPs   590 compared to the QD-loaded polymersome because the outer PEG layer provides a versatile   591 stealth shield for the non-targeted QD-loaded NP, while the conjugation of folate on the   592 nanoparticles surface decreases the shielding effect of the PEG outer layer.

  593

3.9.1. Fluorescence microscopy images of organs

  594

The fluorescence microscopy images of frozen sections of organs extracted at 6 h after   595 injection of QD-NPs, FA-QD-NPs, and free QD nanoprobes are documented in Figure 11.   596 Compared to other organs, the liver and spleen illustrated the maximum fluorescence   597 intensities in free QD–injected mice; meanwhile, the fluorescence intensities were lower for   598 the QD-NP- and FA-QD-NP-injected groups.

  599

The fluorescence intensities in the liver and spleen of FA-QD-NP-injected mice was higher   600 than those in QD-NP-injected mice. This outcome can be ascribed to the reduction of the   601 PEG shielding effect after folate conjugation on the surface of the targeted nanoprobe. In   602 addition, the fluorescence intensities in tumor tissues of mice was as follows: FA-QD-NPs >   603 QD-NPs > free QDs. This was in agreement with the obtained results from quantitative   604 fluorescence assay of organs.

  605

Targeting of QD-NPs to cancer cells by FA further increased their tumor accumulation,   606 showing a remarkable potential of tumor-targeted FA-QD-NPs as co-delivery vehicles for   607 imaging agents along with anticancer drugs. A comparison of in vivo fluorescence analysis   608 after injection of non-targeted and tumor-targeted QD-NPs demonstrated that both passive   609 and active tumor-targeting mechanisms are engaged in QD accumulation within the tumor.

   | P a g e 25  

  610

The EPR effect is likely the phenomenon responsible for the passive targeting of QD-loaded   611 polymersomes to tumors. The active targeting was achieved by conjugation of folate on the   612 surface of QD-loaded polymersomes.

  613

Folate enhances the accumulation of FA-QD-NPs within the tumor and cancer cells, as   614 evidenced by both in vivo and in vitro experiments.

  615

The represented data verified that folate-conjugated biodegradable multimodal NPs intensify   616 the carrier accumulation in the folate receptor–overexpressed 4T1 tumors and their effective   617 internalization by cancer cells.

  618

3.9.2. Evaluation of acute toxicity

  619

In addition to in vitro and in vivo studies, histological analysis was performed on the tissues   620 obtained from the major organs such as the liver, spleen, heart, lungs, and kidneys post-   621 injection to investigate signs of acute FA-QD-NP and QD-NP toxicity. Tissues were   622 harvested 60 days after intravenous injection in the FA-QD-NP and QD-NP BALB/c mice.   623 As illustrated in Figure 12, no signs of toxicity in H&E-stained tissue sections were observed   624 from the animals receiving 10 consecutive intravenous injections of targeted and non-targeted   625 formulations and free QDs (equivalent QD concentration, 18 mg/kg) in comparison with   626 animals treated with 0.9% NaCl solution. Within 60 days of the experiment, no changes in   627 the body weight or drinking and eating behavior of the mice were detected.

  628

To our knowledge, there have been no reports on the long-term toxicities of MSA-capped   629 CdTe QDs. Most studies document the cellular toxicity of CdTe QDs and lower toxicity of   630 CdSe/ZnS [64]. The low toxicity of encapsulated imaging agents is not pivotal for their   631 application in cancer chemotherapy and imaging when the carrier is also encapsulated in the   632 highly toxic anticancer drug along with QDs as a nanoprobe for fluorescence imaging. The   633 presented study demonstrated that MSA-capped CdTe QDs were not toxic in the   634 concentrations used for in vivo experiments; in addition, we did not observe any acute   635

   | P a g e 26  

toxicity upon 10 consecutive intravenous injections of the nanoprobe. We think that the   636 obtained results provide a basis for the possible clinical application of MSA-capped QDs.

  637

All of these results suggest that these functionalized NPs encapsulating QDs can be further   638 improved for specific in vivo applications. Further investigations on these targeted QD-loaded   639 NP formulations are needed to examine how they can be safely excreted from the body.

  640

3.10.

  641

The therapeutic potential of the prepared theranostic formulation

Next, the efficacy of folate-conjugated, QD-DOX-loaded NP formulations was investigated   642 using allograft models of breast cancer developed through subcutaneously injecting 4T1 cells   643 into the flanks of BALB/c female mice. After tumors had developed to 80–100 mm3, the   644 therapeutic efficacies of the prepared targeted and untargeted theranostic formulations were   645 evaluated by dividing the tumor-bearing mice into five groups (n = 5) as a way of minimizing   646 weight and tumor-size differences among the groups. The tumor growth rate in terms of the   647 mean tumor size (mm3) is presented in Figure 13.

  648

Analyzing tumor growth curves demonstrated that a single intravenous injection of either FA-   649 QD-DOX-NPs or QD-DOX-NPs was significantly more efficacious in inhibiting tumor   650 growth compared with injections in the free QD, control, or free DOX group, and this is   651 likely due to enhanced permeation and retention effects. Moreover, the enhanced tumor-   652 inhibition effects of FA-conjugated QD-DOX-NPs compared with non-targeted QD-DOX-   653 NPs might have been due to the targeted particles’ binding to folate receptor proteins on the   654 4T1 cells, thereby possibly delaying clearance and extravasations from the tumor tissue. In   655 fact, folate receptor–targeted NPs are internalized after binding to folate receptor proteins,   656 and higher intracellular DOX transports might enhance cytotoxicity and increase this group’s   657 potency at inhibiting tumor growth. While free DOX was slightly effective in preventing   658 tumor growth compared to 0.9% saline, no significant differences were observed between   659 treatments with saline and free QDs.

  660

   | P a g e 27  

The antitumor efficacy of QD-DOX-NPs at 7 mg/kg was marginal, while the therapeutic   661 efficacy was significantly higher than in the free DOX–treated group.

  662

Survival rates of 4T1-tumor-bearing mice treated with QD-DOX-NPs and free DOX at a dose   663 of 7 mg/kg during a 60-day period after treatment can be seen in Figure 14.

  664

All of the mice treated with 7 mg/kg free DOX died after 40 days. Three mice out of five that   665 receive QD-DOX-NPs remained alive until day 60. Meanwhile, all animals in the FA-QD-   666 DOX-NPs–treated group remains alive until day 60. All mice died after 60 and 55 days in the   667 free QD– and saline-treated groups, respectively. The difference in survival time for the free   668 DOX–treated group compared with the QD-DOX-NP- or FA-DOX-NP-treated groups was   669 statistically significant (ANOVA at a 95% confidence interval), and this is ascribed to free   670 DOX’s high toxicity. In contrast, there was no statistically difference between survival rates   671 in the free DOX–and saline-treated groups.

  672

FA-QD-DOX-NP treatment indicated ideal efficacy, since the tumor growth rate was   673 significantly slower for mice in the group treated with this than it was in other groups   674 (ANOVA at a 95% confidence interval). QD-DOX-NPs were also more efficacious than free   675 DOX, free QDs, and saline treatment (control), but they were significantly less efficacious   676 than FA-QD-DOX-NPs. The obtained results demonstrate that, after just one intravenous   677 administration, FA-QD-DOX-NP inoculation was the most successful treatment for inhibiting   678 4T1 tumor growth in mice.

  679

Previously, heart damage, swollen cardiac muscle fibers, interstitial edema, and inflammatory   680 infiltration were reported after DOX administration [65, 66]. In the current study, as   681 illustrated in Figure 15, in contrast to the free DOX–treatment group, no noticeable changes   682 were observed in the myocardial pathology of the saline- or targeted and non-targeted QD-   683 DOX-loaded NP–treated groups after injection of a therapeutic dosage of DOX that also   684 contained an adequate amount of QDs for in vivo fluorescent imaging. Thus, targeted and   685

   | P a g e 28  

non-targeted QD-DOX-loaded NPs did not exhibit any sign of cardiotoxicity in vivo as   686 compared to free DOX.

  687

Each treatment’s toxicity was further evaluated by investigation of its influence on body   688 weight loss (BWL). At the end of the experiment, no obvious body weight change of the mice   689 in any of the treatment groups was seen except for free DOX–treated group (Figure 16). The   690 body weight–change curve shows weight loss for mice treated with free DOX and QD-DOX-   691 NPs at 3 and 7 days post injection, respectively. The BWL that did arise in the DOX-NP-   692 treated group 7 days post-injection might have been due to the slight toxicity of the   693 formulation’s dosage size; the treated mice began recovering their body weights 10 days after   694 receiving the nanoparticulate dosage form.

  695

The observed loss of body weight 7 days post-injection and subsequent recovery after 10   696 days in the DOX-QD-NP group might represent the PLGA-based formulation release profile,   697 which comprises an initial burst followed by continuous DOX release over time. This release   698 pattern is characteristic of the PLGA controlled-release polymer system, and crucially, allows   699 for DOX presence at the administration site over an extended period. Furthermore, mice   700 treated with the targeted formulation did not show BWL during the experiment, possibly   701 verifying the modified pharmacokinetic properties of the targeted formulations in mice   702 bodies as opposed to the non-targeted formulations.

  703

The obtained results demonstrated that the prepared targeted theranostic NP provides the   704 ideal antitumor efficacy in vivo while exhibiting no cardiac or physiological toxicity. In   705 addition, the aforementioned theranostic system, beyond its potent therapeutic effect,   706 enhanced the QD accumulation in tumor tissue 6 h post-injection for bioimaging applications.   707 Nonetheless, as we proved in the current study, QDs can be carefully tailored to obtain   708 modified pharmacokinetics and exhibit less toxicity for biomedical application. Incidentally,   709

   | P a g e 29  

toxicity concern is still a major issue for QD usage in humans, and further investigation is   710 needed to confirm its safety.

  711

4. Conclusion

  712

In summary, the PEG-PLGA NP–encapsulated hydrophilic QD formulation appears to be an   713 excellent platform for co-loading hydrophobic drug such as DOX in the bilayer of NPs for   714 theranostic applications. These PEG-PLGA NP–encapsulated MSA-capped QDs were readily   715 used for targeting and imaging tumors in animals without causing any toxic effects to the   716 animals.

  717

By using the in vitro and fluorescent organ imaging technique, we demonstrated that the QD-   718 NPs accumulated in the tumor via the EPR effect. Furthermore, the accumulation   719 enhancement of the folate receptor–targeted formulation (FA-QD-NPs) in the tumor was   720 observed. The in vivo results also showed that injections of folate receptor–targeted DOX-   721 QD–loaded NPs could significantly increase the therapeutic efficacy of DOX in the 4T1   722 tumor model in mice. The current study provides crucial information for the future   723 development of QD-DOX NP formulations for early breast cancer detection, therapy, and   724 image-guided surgery applications.

  725

Declaration of Interest

  726

The authors declare that they have no conflicts of interests.

  727   728   729

Acknowledgments

The authors are grateful for the financial support provided by the Mashhad University of   730 Medical Sciences (No. 910040). The authors would also like to thank Prof. Fatemeh Atyabi   731 from the Tehran University of Medical Sciences for kindly providing 4T1 cell line. Also we   732 really appreciate all assistance provided by Dr. Jafarian and Mr. Ghanbari from Ghaem   733 Hospital for histopathological analysis.

  734

   | P a g e 30  

  735

References

1. Howell A, Anderson AS, Clarke RB, Duffy SW, Evans D, Garcia-Closas M, Gescher   736 AJ, Key TJ, Saxton JM, Harvie MN. Risk determination and prevention of breast   737 cancer. Breast Cancer Res. 2014 Sep 28;16(5):446.

  738

2. Bolyard C, Yoo JY, Wang PY, Saini U, Rath KS, Cripe TP, Zhang J, Selvendiran K,   739 Kaur B. Doxorubicin Synergizes with 34.5ENVE to Enhance Antitumor Efficacy   740 against Metastatic Ovarian Cancer. Clin Cancer Res. 2014 Oct 7.

  741

3. Moon H, Kang J, Sim C, Kim J, Lee H, Chang JH, Kim H. Multifunctional   742 theranostic contrast agent for photoacoustics- and ultrasound-based tumor diagnosis   743 and ultrasound-stimulated local tumor therapy. J Control Release. 2015 Oct 9;218:63-   744 71.

  745

4. Zhang M, Wang T, Zhang L, Li L, Wang C. Near-Infrared Light and pH-Responsive   746 Polypyrrole@Polyacrylic acid/Fluorescent Mesoporous Silica Nanoparticles for   747 Imaging and Chemo-Photothermal Cancer Therapy. Chemistry. 2015 Nov   748 2;21(45):16162-71.

  749

5. Chan MH, Lin HM. Preparation and identification of multifunctional mesoporous   750 silica nanoparticles for in vitro and in vivo dual-mode imaging, theranostics, and   751 targeted tracking. Biomaterials. 2015 Apr;46:149-58.

  752

6. Andrade ÂL, Fabris JD, Domingues RZ, Pereira MC. Current Status of Magnetite-   753 Based Core@Shell Structures for Diagnosis and Therapy in Oncology Short running   754 title: Biomedical Applications of Magnetite@Shell Structures. Curr Pharm Des. 2015   755 Sep 16.

  756

7. Liu Y, Li J, Liu F, Feng L, Yu D, Zhang N. Theranostic Polymeric Micelles for the   757 Diagnosis and Treatment of Hepatocellular Carcinoma. J Biomed Nanotechnol. 2015   758 Apr;11(4):613-22.

  759    | P a g e 31  

8. Shao D, Li J, Pan Y, Zhang X, Zheng X, Wang Z, Zhang M, Zhang H, Chen L.   760 Noninvasive theranostic imaging of HSV-TK/GCV suicide gene therapy in liver   761 cancer by folate-targeted quantum dot-based liposomes. Biomater Sci. 2015   762 Jun;3(6):833-41.

  763

9. Sk UH, Kojima C.Dendrimers for theranostic applications. Biomater Sci. 2015   764 Jun;3(6):833-41.

  765

10. Charron DM, Chen J, Zheng G. Theranostic lipid nanoparticles for cancer medicine.   766 Cancer Treat Res. 2015;166:103-27.

  767

11. Sao R, Vaish R, Sinha N. Multifunctional Drug Delivery Systems Using Inorganic   768 Nanomaterials: A Review. J Nanosci Nanotechnol. 2015 Mar;15(3):1960-72.

  769

12. Chiang WH, Huang WC, Chang CW, Shen MY, Shih ZF, Huang YF, Lin SC, Chiu   770 HC. Functionalized polymersomes with outlayered polyelectrolyte gels for potential   771 tumor-targeted delivery of multimodal therapies and MR imaging. J Control Release.   772 2013 Jun 28;168(3):280-8.

  773

13. Qi L, Guo Y, Luan J, Zhang D, Zhao Z, Luan Y. Folate-modified bexarotene-loaded   774 bovine serum albumin nanoparticles as a promising tumor-targeting delivery system.   775 J. Mater. Chem. B. 2014; 2: 8361-8371.

  776

14. Zhao L, Li N, Wang K, Shi C, Zhang L, Luan Y. A review of polypeptide-based   777 polymersomes. Biomaterials. 2014; 35(4):1284-301.

  778

15. Li N, Zhang P, Huang C, Song Y, Garg S, Luan Y. Co-delivery of doxorubicin   779 hydrochloride and verapamil hydrochloride by pH-sensitive polymersomes for the   780 reversal of multidrug resistance. RSC Adv. 2015; 5: 77986-77995.

  781

16. Lai MH, Lee S, Smith CE, Kim K, Kong H. Tailoring polymersome bilayer   782 permeability improves enhanced permeability and retention effect for bioimaging.   783 ACS Appl Mater Interfaces. 2014 Jul 9;6(13):10821-9.

  784

   | P a g e 32  

17. Nahire R, Haldar MK, Paul S, Ambre AH, Meghnani V, Layek B, Katti KS, Gange   785 KN, Singh J, Sarkar K, Mallik S. Multifunctional polymersomes for cytosolic   786 delivery of gemcitabine and doxorubicin to cancer cells. Biomaterials. 2014   787 Aug;35(24):6482-97.

  788

18. Xu J, Zhao Q, Jin Y, Qiu L. High loading of hydrophilic/hydrophobic doxorubicin   789 into polyphosphazene polymersome for breast cancer therapy. Nanomedicine. 2014   790 Feb;10(2):349-58.

  791

19. Kim HO, Kim E, An Y, Choi J, Jang E, Choi EB, Kukreja A, Kim MH, Kang B, Kim   792 DJ, Suh JS, Huh YM, Haam S. A biodegradable polymersome containing Bcl-xL   793 siRNA and doxorubicin as a dual delivery vehicle for a synergistic anticancer effect.   794 Macromol Biosci. 2013 Jun;13(6):745-54.

  795

20. Li S, Byrne B, Welsh J, Palmer AF. Self-assembled poly(butadiene)-b-poly(ethylene   796 oxide) polymersomes as paclitaxel carriers. Biotechnol Prog. 2007 Jan-Feb;23(1):278-   797 85.

  798

21. Upadhyay KK, Bhatt AN, Castro E, Mishra AK, Chuttani K, Dwarakanath BS, Schatz   799 C, Le Meins JF, Misra A, Lecommandoux S. In vitro and in vivo evaluation of   800 docetaxel loaded biodegradable polymersomes. Macromol Biosci. 2010 May   801 14;10(5):503-12.

  802

22. Jain JP, Kumar N. Development of amphotericin B loaded polymersomes based on   803 (PEG)(3)-PLA co-polymers: Factors affecting size and in vitro evaluation. Eur J   804 Pharm Sci. 2010 Aug 11;40(5):456-65.

  805

23. Suk JS, Xu Q, Kim N, Hanes J, Ensign LM. PEGylation as a strategy for improving   806 nanoparticle-based drug and gene delivery. Adv Drug Deliv Rev. 2015 Oct 9.

   | P a g e 33  

  807

24. Wong AD, Ye M, Ulmschneider MB, Searson PC. Quantitative Analysis of the   808 Enhanced Permeation and Retention (EPR) Effect. PLoS One. 2015 May   809 4;10(5):e0123461.

  810

25. Bae YH, Park K. Targeted drug delivery to tumors: Myths, reality and possibility. J   811 Control Release. 2011 August 10; 153(3): 198–205.

  812

26. Hu L, Pang S, Hu Q, Gu D, Kong D, Xiong X, Su J. Enhanced antitumor efficacy of   813 folate targeted nanoparticles co-loaded with docetaxel and curcumin. Biomed   814 Pharmacother. 2015 Oct;75:26-32.

  815

27. Yang Y, Li N, Nie Y, Sheng M, Yue D, Wang G, Tang JZ, Gu Z. Folate-Modified   816 Poly(malic acid) Graft Polymeric Nanoparticles for Targeted Delivery of   817 Doxorubicin: Synthesis, Characterization and Folate Receptor Expressed Cell   818 Specificity. J Biomed Nanotechnol. 2015 Sep;11(9):1628-39.

  819

28. Cai HH, Pi J, Lin X, Li B, Li A, Yang PH, Cai J. Gold nanoprobes-based resonance   820 Rayleigh scattering assay platform: Sensitive cytosensing of breast cancer cells and   821 facile monitoring of folate receptor expression. Biosens Bioelectron. 2015 Dec   822 15;74:165-9.

  823

29. Wu D, Zheng Y, Hu X, Fan Z, Jing X. Anti-tumor activity of folate targeted   824 biodegradable polymer-paclitaxel conjugate micelles on EMT-6 breast cancer model.   825 Mater Sci Eng C Mater Biol Appl. 2015 Aug;53:68-75.

  826

30. Satsangi A, Roy SS, Satsangi RK, Tolcher AW, Vadlamudi RK, Goins B, Ong JL.   827 Synthesis of a novel, sequentially active-targeted drug delivery nanoplatform for   828 breast cancer therapy. Biomaterials. 2015 Aug;59:88-101.

  829

31. Bahrami B, Mohammadnia-Afrouzi M, Bakhshaei P, Yazdani Y, Ghalamfarsa G,   830 Yousefi M, Sadreddini S, Jadidi-Niaragh F, Hojjat-Farsangi M. Folate-conjugated   831

   | P a g e 34  

nanoparticles as a potent therapeutic approach in targeted cancer therapy. Tumour   832 Biol. 2015 Aug;36(8):5727-42.

  833

32. Frasco MF, Chaniotakis N. Bioconjugated quantum dots as fluorescent probes for   834 bioanalytical applications. Anal Bioanal Chem. 2010; 396(1): 229-40.

  835

33. Nash CH 3rd, Jones SE, Moon TE, Davis SL, Salmon SE. Prediction of outcome in   836 metastatic breast cancer treated with adriamycin combination chemotherapy. Cancer.   837 1980 Dec 1;46(11):2380-8.

  838

34. Wen CJ, Zhang LW, Al-Suwayeh SA, Yen TC, Fang JY. Theranostic liposomes   839 loaded with quantum dots and apomorphine for brain targeting and bioimaging. Int J   840 Nanomedicine. 2012;7:1599-611.

  841

35. Li JM, Wang YY, Zhao MX, Tan CP, Li YQ, Le XY, Ji LN, Mao ZW.   842 Multifunctional QD-based co-delivery of siRNA and doxorubicin to HeLa cells for   843 reversal of multidrug resistance and real-time tracking. Biomaterials. 2012   844 Mar;33(9):2780-90.

  845

36. Alibolandi M, Ramezani M, Abnous K, Sadeghi F, Atyabi F, Asouri M, Ahmadi AA,   846 Hadizadeh F. In vitro and in vivo evaluation of therapy targeting epithelial-cell   847 adhesion-molecule aptamers for non-small cell lung cancer. J Control Release. 2015   848 Jul 10;209:88-100.

  849

37. B. Jeong, Y. K. Choi, Y. H. Bae, G. Zentner, S. W. Kim. New biodegradable   850 polymers for injectable drug delivery systems. J Control Release. 62 (1999) 109-14.

  851

38. Alibolandi M, Abnous K, Ramezani M, Hosseinkhani H, Hadizadeh F. Synthesis of   852 AS1411-aptamer-conjugated CdTe quantum dots with high fluorescence strength for   853 probe labeling tumor cells. J Fluoresc. 2014 Sep;24(5):1519-29.

  854

39. Pillai JJ, Thulasidasan AKT, Anto RJ, Devika NC, Ashwanikumar N,

Kumar   855

GSV. Curcumin entrapped folic acid conjugated PLGA–PEG nanoparticles exhibit   856

   | P a g e 35  

enhanced anticancer activity by site specific delivery. RSC Adv. 2015; 5: 25518-   857 25524

  858

40. Su CW, Chiang CS, Li WM, Hu SH, Chen SY. Multifunctional nanocarriers for   859 simultaneous encapsulation of hydrophobic and hydrophilic drugs in cancer treatment.   860 Nanomedicine (Lond). 2014 Jul;9(10):1499-515.

  861

41. Yang X, Wang D, Ma Y, Zhao Q, Fallon JK, Liu D, Xu XE, Wang Y, He Z, Liu F.   862 Theranostic nanoemulsions: codelivery of hydrophobic drug and hydrophilic imaging   863 probe for cancer therapy and imaging. Nanomedicine (Lond). 2014 Dec;9(18):2773-   864 85.

  865

42. Wang H, Agarwal P, Zhao S, Xu RX, Yu J, Lu X, He X. Hyaluronic acid-decorated   866 dual responsive nanoparticles of Pluronic F127, PLGA, and chitosan for targeted co-   867 delivery of doxorubicin and irinotecan to eliminate cancer stem-like cells.   868 Biomaterials. 2015 Dec;72:74-89.

  869

43. Alibolandi M, Ramezani M, Sadeghi F, Abnous K, Hadizadeh F. Epithelial cell   870 adhesion molecule aptamer conjugated PEG-PLGA nanopolymersomes for targeted   871 delivery of doxorubicin to human breast adenocarcinoma cell line in vitro. Int J   872 Pharm. 2015 Feb 1;479(1):241-51.

  873

44. Discher BM, Won YY, Ege DS, Lee JCM, Bates FS, Discher DE, Hammer DA.   874 Polymersomes: Tough vesicles made from diblock copolymers. Science 1999;   875 284(5417):1143–1146.

  876

45. Discher DE, Eisenberg A. Polymer vesicles. Science 2002; 297: 967–973.

  877

46. Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL:   878 Hydrolysis-triggered controlled release vesicles. J Controlled Release 2004; 96: 37–   879 53.

  880

   | P a g e 36  

47. Debets MF, Leenders WP, Verrijp K, Zonjee M, Meeuwissen SA, Otte-Höller I, van   881 Hest JC. Nanobody-functionalized polymersomes for tumor-vessel targeting.   882 Macromol Biosci. 2013 Jul;13(7):938-45.

  883

48. Chiang WH, Huang WC, Chang CW, Shen MY, Shih ZF, Huang YF, Lin SC, Chiu   884 HC. Functionalized polymersomes with outlayered polyelectrolyte gels for potential   885 tumor-targeted delivery of multimodal therapies and MR imaging. J Control Release.   886 2013 Jun 28;168(3):280-8.

  887

49. Meng F, Engbers GH, Feijen J. Biodegradable polymersomes as a basis for artificial   888 cells: encapsulation, release and targeting. J Control Release. 2005 Jan 3;101(1-   889 3):187-98.

  890

50. Kobayashi H, Watanabe R, Choyke PL. Improving conventional enhanced   891 permeability and retention (EPR) effects; what is the appropriate target? Theranostics.   892 2013 Dec 11;4(1):81-9.

  893

51. Jiang S, Gong X, Zhao X, Zu Y. Preparation, characterization, and antitumor   894 activities of folate-decorated docetaxel-loaded human serum albumin nanoparticles.   895 Drug Deliv. 2015 Feb;22(2):206-13.

  896

52. Alibolandi M, Sadeghi F, Abnous K, Atyabi F, Ramezani M, Hadizadeh F. The   897 chemotherapeutic potential of doxorubicin-loaded PEG-b-PLGA nanopolymersomes   898 in mouse breast cancer model. Eur J Pharm Biopharm. 2015 Aug;94:521-31.

  899

53. Misra R, Sahoo SK. Intracellular trafficking of nuclear localization signal conjugated   900 nanoparticles for cancer therapy. Eur J Pharm Sci 2010; 39: 152–163.

  901

54. Mattsson W, Borgström S, Landberg T. An effective low-dose adriamycin regimen as   902 secondary chemotherapy for metastatic breast cancer patients. Cancer. 1980 Aug   903 1;46(3):433-7.

  904

   | P a g e 37  

55. Magenheim B, Levy MY, Benita S. A new in vitro technique for evaluation of drug   905 release profile from colloidal carriers-ultrafiltration technique at low pressure. Int J   906 Pharm 1993; 94: 115–123.

  907

56. Das M, Sahoo SK. Folate decorated dual drug loaded nanoparticle: role of curcumin   908 in enhancing therapeutic potential of nutlin-3a by reversing multidrug resistance.   909 PLoS One. 2012;7(3):e32920.

  910

57. Wang Y, Yu L, Han L, Sha X, Fang X. Difunctional Pluronic copolymer micelles for   911 paclitaxel delivery: synergistic effect of folate-mediated targeting and Pluronic-   912 mediated overcoming multidrug resistance in tumor cell lines. Int J Pharm 2007; 337:   913 63–73.

  914

58. Sahoo SK, Labhasetwar V. Enhanced antiproliferative activity of transferrin-   915 conjugated paclitaxel-loaded nanoparticles is mediated via sustained intracellular drug   916 retention. Mol Pharm 2005; 2: 373–383.

  917

59. Lin G, Wang X, Yin F, Yong KT. Passive tumor targeting and imaging by using   918 mercaptosuccinic acid-coated near-infrared quantum dots. Int J Nanomedicine. 2015   919 Jan 6;10:335-45.

  920

60. Winnik FM, Maysinger D. Quantum dot cytotoxicity and ways to reduce it. Acc   921 Chem Res. 2013 Mar 19;46(3):672-80.

  922

61. Yong KT, Law WC, Hu R, Ye L, Liu L, Swihart MT, Prasad PN. Nanotoxicity   923 ssessment of quantum dots: from cellular to primate studies. Chem Soc Rev. 2013 Feb   924 7;42(3):1236-50.

  925

62. Maeda H. The enhanced permeability and retention (EPR) effect in tumor vasculature:   926 the key role of tumor-selective macromolecular drug targeting. Advances in Enzyme   927 Regulation. 2001; 41: 189–207.

  928

   | P a g e 38  

63. Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the   929 EPR effect in macromolecular therapeutics: a review. J Control Release 2000; 65:   930 271-84.

  931

64. Pelley JL, Daar AS, Saner MA. State of academic knowledge on toxicity and   932 biological fate of quantum dots. Toxicol. Sci. 2009; 112: 276–296.

  933

65. Li K, Sung RY, Huang WZ, Yang M, Pong NH, Lee SM, Chan WY, Zhao H, To MY,   934 Fok TF, Li CK, Wong YO, Ng PC. Thrombopoietin protects against in vitro and in   935 vivo cardiotoxicity induced by doxorubicin. Circulation 2006; 113: 2211-20.

  936

66. Rahman AM, Yusuf SW, Ewer MS. Anthracycline-induced cardiotoxicity and the   937 cardiac-sparing effect of liposomal formulation. Int J Nanomedicine 2007; 2: 567–   938 583.

  939

67. Alberts DS, Muggia FM, Carmichael J, Winer EP, Jahanzeb M, Venook AP, Skubitz   940 KM, Rivera E, Sparano JA, DiBella NJ, Stewart SJ, Kavanagh JJ, Gabizon AA.   941 Efficacy and safety of liposomal anthracyclines in phase I/II clinical trials. Semin   942 Oncol 2004; 31: 53-90.

  943   944

   | P a g e 39  

  945

Figure legends

Figure 1. Synthesis of (a) PLGA-PEG (conjugation of NH2-PEG-COOH to COOH-PLGA),   946 (b) aminated folic acid (conjugation of ethylene diamine to folic acid), (c) PLGA-PEG-NHS   947 (activation of carboxylic acid of PLGA-PEG-COOH and preparation of PLGA-PEG-NHS)   948 and (d) PLGA-PEG-FA (conjugation of PLGA-PEG-NHS to aminated folic acid).

  949

Figure 2. FTIR spectra of (A) PEG-PLGA and (B) FA-PEG-PLGA copolymer. Red arrow   950 demonstrates the very small peaks of amide bond in FA-PEG-PLGA which is absent in PEG-   951 PLGA.

  952

Figure 3. 1H-NMR spectra of (a) PEG-PLGA, (b) FA-PEG-PLGA and (c) resonances of folic

  953

acid is enlarged for clarity.

  954

Figure 4. High resolution TEM of (A) QD-DOX-NPs and (B) FA-QD-DOX-NPs.

  955

Figure 5. (A) Fluorscence emission spectra of blank polymersome, free QDs and QDs loaded   956 nanopolymersome (λex=360 nm). (B) Free QDs and QDs-loaded nanopolymersome under   957 UV-irradiation (λex= 365 nm). High resolution TEM image of (C) MSA-capped CdTe QDs   958 and (D) QDs-loaded nanopolymersome.

  959

Figure 6. Release profile of DOX-NPs and QD-DOX-NPs in PBS pH 7.4, 37°C.

  960

Figure 7. Cellular uptake of FA-QD-DOX-NP (A); QD-DOX-NP (B); free DOX/QD (C)   961 and free folic acid and FA-QD-DOX-NPs (D) treated 4T1 cell and cellular uptake FA-QD-   962 DOX-NP (E); QD-DOX-NP (F); free DOX/QD (G) and free folic acid and FA-QD-DOX-   963 NPs (H) treated MCF-7 cell.

  964

Figure 8. Cytotoxicity of free DOX, QD-DOX-loaded nanopolymersomes; folate conjugated   965 QD-DOX-loaded nanopolymersomes on the 4T1 (A) and MCF-7 (B) cell lines after 48 h (n =   966 4, error bars represent standard deviation).

  967

   | P a g e 40  

Figure 9. Cytotoxicity of free QDs, QD-loaded nanopolymersomes; folate conjugated QD-   968 loaded nanopolymersomes on the 4T1 (A) and MCF-7 (B) cell lines after 48 h (n = 4, error   969 bars represent standard deviation).

  970

Figure 10. Systemic distribution of free quantum dot, QD-loaded nanopolymersome and   971 folate conjugated QD-loaded nanopolymersome in (A) different organs and (B) tumors; 6   972 hours post intravounsly injection of nanoprobe.

  973

Figure 11. Fluorescence microscopy images of frozen sections of organs extracted at 6 hrs   974 after injection of QD-NPs, FA-QD-NPs and free QD nanoprobes.

  975

Figure 12. In vivo acute toxicity after administration of ten consecutive injections of free   976 QDs, QD-loaded nanopolymersome and folic acid conjugated QD-loaded nanopolymersome   977 at a QD concentration of 18 mg/kg body weight during 60 days (every 6 days).

  978

Figure 13. Tumor growth inhibitory efficacy of free DOX, QD-DOX-NP and FA-QD-DOX-   979 NP groups in comparison with saline and free QD groups in the subcutaneous mouse model   980 of 4T1 (n = 5, error bars represent standard deviation).

  981

Figure 14. Kaplan-Meier survihjikval curves of subcutaneous mouse model of 4T1 treated   982 with DOX, DOX-QD-NP, FA-QD-DOX-NP, free DOX, free QD and saline.

  983

Figure 15. Mouse myocardium: treated with free DOX, DOX-QD-NP or FA-QD-DOX-NP   984 (DOX concentration of 7 mg/kg body weight (H&E stain, magnification: 40×). (A: Free   985 DOX; B: QD-DOX-NPs; C: FA-QD-DOX-NPs treated; D: Saline).

  986

Figure 16. Body weight (g) of 4T1 tumor-bearing mice (n = 5, error bars represent standard   987 deviation) treated with QD-DOX-NPs, FA-QD-DOX-NPs, free DOX and free QD (DOX: 7   988 mg/kg, QD: 18 mg/kg).

  989   990   991   992

   | P a g e 41  

  993

Fig 1

  994   995

  996

Fig 2

  997   998    | P a g e 42  

  999

Fig 3

  1000   1001   1002   1003   1004

  1005

Fig 4

  1006    | P a g e 43  

  1007

Fig 5

  1008   1009

   | P a g e 44  

  1010

Fig 6

  1011   1012

  1013

Fig 7

  1014   1015

   | P a g e 45  

  1016

Fig 8

  1017   1018   1019

  1020

Fig 9

  1021   1022

   | P a g e 46  

  1023

Fig 10

  1024   1025

  1026

Fig 11

  1027   1028   1029

   | P a g e 47  

  1030

Fig 12

  1031   1032

  1033

Fig 13

  1034   1035

   | P a g e 48  

  1036

Fig 14

  1037   1038   1039

  1040

Fig 15

  1041    | P a g e 49  

  1042

Fig 16

  1043   1044

   | P a g e 50  

Table 1. Characteristics of the prepared nanopolymersome formulations.

  1045

PEGFormulation Blank NPs Blank FA-NPs QD-NPs FA-QD-NPs DOX-NPs FA-DOX-NPs QD-DOX-NPs FA-QD-DOX-NPs

PLGA

DOX:QD

DOX: EE%

DOX: LC%

(mg)

(mg):(mg)

QD: EE%

QD: LC%

20

0:0

20 20 20 20 20 20 20

0:0 0:4 0:4 1:0 1:0 1:4 1:4

------------

------------

------------

------------

------------

------------

------------

------------

------------

------------

53.21±2.41

10.64±0.34

------------

------------

51.67±4.21

10.33±0.48

86.51±3.68

4.32±0.65

-------------

------------

84.75±0.98

4.53±0.21

------------

------------

52.87±5.52

10.57±0.33

83.42±6.43

4.16±0.19

54.26±1.23

10.82±0.87

84.98±5.65

4.19±0.63

Size (nm)

PDI

156.21±0.21

0.13

152.5±3.9 154.76±1.36

0.121

151.62±2.52

0.21

166.03±4.21

0.17

165.89±4.21

0.25

167.32±4.13

0.181

170.53±1.21

0.16

  1046   1047   1048

   | P a g e 51