Antitumor efficacy and cardiotoxic effect of doxorubicin-loaded mixed micelles in 4T1 murine breast cancer model. Comparative studies using Doxil® and free doxorubicin

Journal Pre-proof Antitumor efficacy and cardiotoxic effect of doxorubicin-loaded mixed micelles in 4T1 murine breast cancer model. Comparative studies using Doxil® and free doxorubicin Maximiliano Cagel, Marcela A. Moretton, Ezequiel Bernabeu, Marcela Zubillaga, Eduardo Lagomarsino, Silvia Vanzulli, Melisa B. Nicoud, Vanina A. Medina, Maria J. Salgueiro, Diego A. Chiappetta PII:

S1773-2247(19)31409-1

DOI:

https://doi.org/10.1016/j.jddst.2020.101506

Reference:

JDDST 101506

To appear in:

Journal of Drug Delivery Science and Technology

Received Date: 16 September 2019 Revised Date:

4 December 2019

Accepted Date: 5 January 2020

Please cite this article as: M. Cagel, M.A. Moretton, E. Bernabeu, M. Zubillaga, E. Lagomarsino, S. Vanzulli, M.B. Nicoud, V.A. Medina, M.J. Salgueiro, D.A. Chiappetta, Antitumor efficacy and cardiotoxic effect of doxorubicin-loaded mixed micelles in 4T1 murine breast cancer model. Comparative studies using Doxil® and free doxorubicin, Journal of Drug Delivery Science and Technology (2020), doi: https:// doi.org/10.1016/j.jddst.2020.101506. 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. © 2020 Published by Elsevier B.V.

Author contribution statement Maximiliano Cagel: Formal analysis, Investigation, Writing - Original Draft Marcela A. Moretton: Conceptualization, Methodology, Formal analysis, Writing - Original Draft, Writing - Review & Editing Ezequiel Bernabeu: Investigation, Formal analysis Marcela Zubillaga: Resources, Writing - Review & Editing Eduardo Lagomarsino: Resources, Formal analysis Silvia Vanzulli: Investigation, Formal analysis Melisa B. Nicoud: Investigation, Formal analysis Vanina A. Medina: Investigation, Resources, Writing - Review & Editing Maria J. Salgueiro: Conceptualization, Methodology, Formal analysis, Resources, Writing - Review & Editing Diego A. Chiappetta: Conceptualization, Methodology, Formal analysis, Resources, Writing - Original Draft, Writing - Review & Editing

Antitumor efficacy and cardiotoxic effect of doxorubicin-loaded mixed micelles in 4T1 murine breast cancer model. Comparative studies using Doxil® and free doxorubicin Maximiliano Cagela,f, Marcela A. Morettona,f,*, Ezequiel Bernabeua,f, Marcela Zubillagab,f, Eduardo Lagomarsinoc, Silvia Vanzullid, Melisa B. Nicoude, Vanina A. Medinae, Maria J. Salgueirob, Diego A. Chiappettaa,f a

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de

Tecnología Farmacéutica I, Buenos Aires, Argentina. b

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de

Física, Buenos Aires, Argentina. c

Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Cátedra de

Atención Farmacéutica y Farmacia Clínica, Buenos Aires, Argentina. d

Departamento de Patología. Instituto de Investigaciones Hematológicas

(IIHEMA), Academia Nacional de Medicina. e

Laboratorio de Biología Tumoral e Inflamación. Instituto de Investigaciones

Biomédicas (BIOMED), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Católica Argentina (UCA). f

National Science Research Council (CONICET), Buenos Aires, Argentina.

*Corresponding author Dr. Marcela A. Moretton Departamento de Tecnología Farmacéutica. Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. 956 Junín St., 6th Floor, Buenos Aires CP1113, Argentina Email: [email protected] Phone/Fax: +54-11-5287-4633

Abstract Doxorubicin-loaded mixed micelles (DOX-micelles) were formulated employing TPGS and a poloxamine (Tetronic® T1107) in order to investigate their pharmaceutical application as an alternative anticancer nanotherapy. DOXmicelles presented unimodal behaviour, exhibiting average size values of 10.7 ± 0.2 nm and 10.4 ± 0.1 nm, before and after lyophilization, respectively. The in vitro cytotoxic effect of DOX-micelles was significantly higher (p < 0.05) than Doxil® on 4T1 murine breast cancer cells. The in vitro cell-based internalization assays showed a significant augment (p < 0.05) of the intracellular DOX content for the DOX-micelles in comparison to the liposomes and free DOX in these 4T1 cells at 2 and 6 h. Besides, DOX-micelles presented higher in vivo anticancer effect than Doxil® and

equivalent

potency

to

the

free

drug. Moreover,

the in

vivo histopathological toxicity studies revealed that the DOX-micelles produced significant

less

cardiac

damage

than

free

DOX,

while

the in

vivo immunohistochemical assays exhibited a significantly higher DNA damage in the cardiac tissue with the free DOX than with our DOX-micelles. All these results demonstrate that our novel nanotechnological platform could successfully deliver DOX to the tumor with reduced cardiotoxicity, thus showing promising clinical potential as an anticancer drug delivery carrier.

Key words: doxorubicin; mixed micelles; TPGS; Doxil®; 4T1 breast cancer model.

1. Introduction Cancer accounts for 9.6 million deaths in 2018, appearing as the second leading cause of death globally [1]. In particular, breast cancer (BC) emerges as the most frequent cancer among women and the second most common cancer worldwide. Overall, it occupies the fifth position in causes of death from cancer [2]. In this sense, doxorubicin (DOX) is widely employed as a first-line medicine in the BC therapy, being one the most potent chemotherapeutic agents. However, it presents numerous

adverse

effects

that

limit

its

administered

dose,

such

as

myelosuppression, hepatotoxicity and life-threatening cardiotoxicity, mainly due to its non-specific distribution and relatively short half-life, which suggests a rapid tissue uptake [3]. Aiming for the improvement of both the toxicity profile and the efficacy of the therapy, several DOX-loaded nanotechnological platforms have been studied as potential drug-delivery systems. Within this framework, the Food and Drug Administration (FDA) has approved a liposomal formulation known as Doxil® in the United States of America (USA) and Caelyx® in Europe for the treatment of AIDS-related Kaposi’s sarcoma (1995), recurrent ovarian cancer (1998) and metastatic breast cancer (MBC) (2003) [4]. In a multicentre phase III clinical study, patients with MBC were administered with schemes of Doxil® 50 mg/m2 every 4 weeks, demonstrating a better safety profile than schemes of free DOX 60 mg/m2 every 3 weeks, with fewer cases of alopecia, nausea, myelosuppression and lower risk of congestive heart failure and cardiac events. However, parameters like overall survival, response rates and median duration of response were comparable, suggesting that Doxil® did not improve the efficacy of

the free drug [5]. Moreover, it has been studied that the “PEGylation” process strongly affects the cellular internalization, thus affecting the drug cellular uptake of these liposomes [6,7]. Among nanocarriers, one of the most promising strategies for improving the solubility and stability of hydrophobic drugs, increasing the cellular uptake and enhancing in vivo parameters, are the polymeric single micelles [8,9]. In the past few years, an interesting approach to achieving improvements in their kinetic, thermodynamic and functionalization properties, has been the mixture of two or more block co-polymers to obtain “polymeric mixed micelles” [10,11]. In our previous study, a DOX-loaded mixed micellar preparation with excellent colloidal properties was formulated and characterized. It was comprised of the poloxamine Tetronic® T1107 and an amphiphilic water-soluble derivative of natural source vitamin E and polyethylene glycol (PEG), known as D-α-tocopheryl PEG 1000 succinate (TPGS). The mixed micellar system has shown significantly higher in vitro cytotoxicity than Doxil® in a BC cell line (MDA-MB-231) and has proved to be more cytotoxic than both Doxil® and free DOX against an ovarian cancer cell line (SKOV-3). Besides, the mixed micellar formulation presented a significantly increased in vitro intracellular DOX content against MDA-MB-231 cells, when compared to Doxil® and a significantly higher in vitro cellular uptake against SKOV3 cells, as compared to both Doxil® and free DOX [12]. In a recent study, we have compared the in vivo toxicity profile of our DOX-loaded mixed micellar formulation with the ones of Doxil® and free DOX, in a zebrafish (Danio rerio) model. The results showed that the DOX-loading process within

mixed micelles reduced the toxic effects of the free drug, as the DOX-loaded mixed micellar system was less cardiotoxic, produced less morphological alterations in the zebrafish and showed diminished neurotoxic effects, when compared to the DOX solution [13]. Considering our previous results, the objective of the present work was to study both the antitumor efficacy and the cardiotoxic effect of DOX-loaded TPGS:T1107 micelles, in comparison with Doxil® and free DOX, in a 4T1 murine BC model developed in BALB/c mice. The in vitro cytotoxicity and cellular uptake were evaluated in 4T1 BC cells. Furthermore, the in vivo antitumor effect and cardiac toxicity assays were carried out in 4T1-tumor bearing mice. 2. Materials and Methods 2.1 Materials Doxorubicin hydrochloride (99.9%) was purchased from LKM Laboratories (Argentina). Tetronic® 1107 (T1107, MW ~15.0 kDa, 70 wt% PEO) was a gift of BASF (Argentina). TPGS (MW ~1513 g/mol) was purchased from Eastman Chemical Company (USA) and sodium deoxycholate (NaDC) was purchased from Riedel-de Häen® (Germany). Doxil® was purchased from Raffo Laboratories (Argentina). CellTiter-Blue® Reagent (Promega). All solvents were analytical or high performance liquid chromatography (HPLC) grade and were used following the manufacturer’s instructions. 2.2 Preparation of blank mixed micelles

The mixed micellar formulation composed of T1107 and TPGS (1:3 polymer weight ratio, 2% w/v total polymer concentration) was prepared as we previously described [12]. Each polymer was equilibrated overnight (25 °C) in distilled water before use. 2.3 Preparation of DOX/NaDC complex-loaded micellar system 2.3.1 Complex preparation We have previously demonstrated that the hydrophobic complex of DOX and sodium deoxycholate (DOX/NaDC) was an excellent candidate to be encapsulated in polymeric mixed micelles [12]. Briefly, DOX hydrochloride (50 mg) was solubilised 10 mL of distilled water (5 mg/mL). Then, NaDC (90 mg) was incorporated to the previous solution, in order to obtain the water-insoluble DOXNaDC complex under magnetic stirring during 2 h. Afterwards, the suspension (10 mL) was centrifuged (20°C, 5600 rpm for 10 min) employing a refrigerated centrifuge (Combi 514R, Hanil Science Industrial Co., Korea). Finally, the precipitate was re-suspended in distilled water (10 mL) and homogenized (Vortex Mixer Wizard, Velp Scientifica, Italy) before use. 2.3.2 DOX/NaDC complex-loaded mixed micelles In a first step, an excess of the DOX-NaDC complex suspension (10 mL) was incorporated into an equal volume (10 mL) of the mixed micellar formulation (4% w/v total polymer concentration) under magnetic stirring for 2 h. Next, the excess of the water-insoluble complex was removed by filtration (0.45 µm acetate cellulose

filters, Microclar, Argentina). Finally, samples were refrigerated (4 °C) or freezedried (see section 2.4) before use. Finally, an UV-visible spectrophotometry method was employed for the quantification of the DOX content at 25 °C. Briefly, DOX solutions were prepared in N,N-Dimethylformamide (25 °C) and the linearity range was assessed in the 5 - 63 µg/mL

range

(λ:

500

nm,

R2:

0.9986,

UV-260,

UV-Visible

Recorder

Spectrophotometer, Shimadzu, Japan). An aqueous NaDC solution and DOX-free micellar dispersions were employed as controls. Assays were done by triplicate and the results were expressed as the mean ± standard deviation (S.D.). From now on, DOX-NaDC complex loaded mixed micelles (2% w/v final polymer concentration, DOX 2 mg/mL) will be referred as DOX-micelles and DOX-free micelles as blank micelles (2% w/v final polymer concentration). 2.4 Measurement of the micellar size, zeta potential and morphological characterization Micellar size (Dh) and size distribution (polydispersity index, PDI) of the DOXmicelles and blank micelles were determined by dynamic light scattering (DLS, Zetasizer Nano-ZSP, Malvern Instruments, United Kingdom). The scattering angle employed was 173° to the incident beam at 25 ºC. Before each determination, every micellar formulation was equilibrated at each temperature for at least 5 min. The results were expressed as the average of three measurements ± S.D. The zeta potential values of these formulations were assessed employing the same

analyzer at 25 °C. Results were expressed as the average of three independent formulations. Additionally, DOX-micelles (2 mL) were introduced in glass vials (5 mL) and frozen at -20 °C overnight. Then, they were lyophilized (freeze-dryer FIC-L05, FIC, Scientific Instrumental Manufacturing, Argentina) for 24 h (freeze-dryer shelf: -14 °C; condenser: -40 °C; pressure: 0.03 mbar). Afterwards, the lyophilizates were resuspended with distilled water, and size, size distribution and zeta potential at 25 °C, were analyzed as described above. Also, DOX-micelles before and after freeze-drying process were analyzed by transmission electron microscopy (TEM, Philips CM-12 TEM apparatus, FEI Company, The Netherlands). In a first step, 5 µL of every sample were covered with a Fomvar film after the aliquots were placed in clean grids. Then, 5 µL of a phosphotungstic acid (1% w/v) solution were employed to negatively stain each sample. Finally, samples washed with 5 µL of distilled water and dried (silicagel container) before the analysis. Additionally, in order to simulate the nanomedicines dilution after an intravenous (i.v.) administration, DOX-micelles and Doxil® were diluted (1/50) with PBS to a final concentration that mimics the formulation dilution upon i.v. administration in mice [14]. The size and size distribution of both nanosystems were measured at 37 °C by DLS as previously described. 2.5 In vitro cytotoxicity assay

The CellTiter-Blue® (Promega) assay was performed as an indicator of cell viability. For this, 4T1 cells seeded in a 96-well plate (2.5 x 103 cells/well) at a final volume of 100 µL and treated with free DOX, Doxil®, DOX-micelles and blank micelles for 48 h. Culture medium was employed to dilute every sample in order to obtain a DOX equivalent concentration range between 0.005 to 1 µg/mL. Afterwards, CellTiter-Blue® Reagent (10 µL/well) was added and cells were incubated for additional 1 h. The fluorescence of resorufin was determined using a NovoStar microplate reader (BMG Labtech). Fluorescence (560(20)Ex/590(10)Em) was determined for 5 replicates per treatment condition. The concentrationdependent cell survival curves were employed to calculate the DOX concentrations required to kill the 50% of the cells (IC50). All values were expressed as percent of untreated control cells set as 100%. One-way ANOVA test (p<0.05) was used for the statistical analysis. 2.6 In vitro determination of apoptosis Flow cytometry was selected to determine the exposure of phosphatidylserine on the apoptotic breast cancer 4T1 cells surface. For this purpose, PI (50 µg/mL) and Annexin V-FITC (BD biosciences, USA) were used for cell staining. A BD AccuriCSampler software (Becton Dickinson Co., USA) was employed to analyze all data. Statistical analysis was made by ANOVA followed by Newman–Keuls' Multiple Comparison Test using GraphPad Prism Version 5.00 software (San Diego, CA, USA). P-values < 0.05 were considered statistically significant. 2.7 In vitro cellular uptake

Breast cancer cells (4T1) at a density of 4 x 105 cells/well were placed in 6-well plates and incubated (37 °C, 5% CO2) for 24 h Next, three formulations (Doxil®, a drug free solution and the DOX-micelles at 100 µg/mL of DOX) were incubated with the tumor cells. Controls were provided by the untreated cells. At different time-points (0.5, 2 and 6 h), the DOX cell uptake was finished by washing the cells with cold PBS (1.5 mL). Afterwards, cells were washed with PBS and a trypsin PBS solution (2.5 µg/mL, 0.25 mL) was added. Finally, lysates were centrifuged (13,000 rpm, 10 min, MiniSpin® plus™, Eppendorf, Germany) and the drug quantification in the supernatants was performed by reverse high performance liquid chromatography (RP-HPLC). Briefly, the analytical method involved a C18 column (4.6 mm×250 mm, 3.5 µm, Xterra RP18, Waters, Ireland) with a mobile phase composed of acetonitrile and water (30:70, v/v; pH: 3 adjusted with phosphoric acid). The flow rate was maintained at 0.6 mL/min at 25 °C. The injection volume was10 µL and the detection wavelength was 233 nm. A BCA protein assay kit (Pierce Corporation, China) was employed to determine the protein content of every sample. Then, the DOX concentrations determined by RP-HPLC were normalized by their protein content. One-way ANOVA test (p < 0.05) (n=3 ± S.D.) was used for the statistical analysis. 2.8 Animals and treatments For in vivo studies, female immunocompetent BALB/c mice (body weight 18–20 g, 6-8 weeks old) were supplied by the Animal Experimental Unit, Faculty of Pharmacy and Biochemistry, University of Buenos Aires. Animals were kept in a 12-h light-dark cycle and free access to food and water. All procedures were

reviewed and approved by the Institutional Committee for the Care and Use of Laboratory Animals, School of Pharmacy and Biochemistry (approval number: 671/2018), and are in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978) and the ARRIVE guidelines for reporting experiments involving animals. Tumors were induced by subcutaneous (s.c.) injection of the (4T1) cells (1 x 105) into the right upper mammary fat pad of female BALB/c mice. When the tumors reached about 100 mm3, the animals were randomly separated into 4 groups (n = 4 each) and intravenously injected with saline (control group), DOX solution, Doxil® and DOX-micelles at a daily dose of 2 mg/kg DOX equivalent on days 0, 3 and 6, respectively. Considering that Doxil® is already a sterile formulation, the rest of the used samples were sterilized by filtration, employing 0.22 µm acetate cellulose filters. Body weight and tumor volumes were monitored and recorded every 2 days. Tumor volumes were calculated according to the formula: (L x W 2) / 2, where L is the longest and W is the shortest tumor diameter (mm), measured with a caliper. For ethical reasons, the animals were monitored according to the Canadian Council on Animal Care and the Guidance Document on the Recognition, Assessment, and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation during the whole treatment [15,16]. Tumor growth data were expressed as tumor volume and relative tumor weight (tumor weight measured with respect to the control’s tumor weight). All data shown are mean ± S.D. Statistical evaluations were made by analysis of variance that was

followed by Tukey Test using GraphPad Prism Version 5.00 software (San Diego, CA, USA). P-values < 0.05 were considered statistically significant. 2.9 Histopathological and immunohistochemical studies Firstly, neutral buffered formalin (10%) was employed to fix all those tissues and tumors that were removed from the animals. Then, every specimen was cut into serial sections (4 µm), after their embedding in paraffin. The hematoxylin-eosin staining (H&E) assay was used to evaluate the histopathological characteristics. Immunohistochemistry assays involved the incubation of the tissues (after blocking) with mouse anti-proliferating cell nuclear antigen (PCNA, 1:100, DAKO Cytomation, Denmark) or rabbit anti-phosphorylated histone H2AX antibody (γH2AX, Cell Signaling Technology, USA) overnight in a humidified chamber at 4 ºC. A Vectastain ABC Kit (Vector Laboratories INC., USA) was employed to detect immunoreactivity and visualized by diamino-benzidine staining (Sigma Chemical Co.). Apoptotic cells were determined by TdT-mediated UTP-biotin Nick End labeling (TUNEL) assay (CHEMICON International, CA, USA) and the visualization of the cells was assessed using an Axiolab Carl Zeiss microscope (Göttingen, Germany) with a Canon PowerShot G5 camera (Japan). Specimens were assessed and scored to provide a quantitative measurement by using ImageJ, NIH software. 2.10 Evaluation of TBARS levels The lipid peroxidation screening was performed by the thiobarbituric acid reactive species (TBARS) assay. In this investigation, the TBARS method employed was

adapted from Martinel Lamas et al., 2013 [17], considering a molar extinction coefficient of ε = 1.56 × 105 /M/cm. Statistical analysis was made by ANOVA followed by Newman–Keuls' Multiple Comparison Test using GraphPad Prism Version 5.00 software (San Diego, CA, USA). P-values < 0.05 were considered statistically significant.

3. Results and discussion 3.1 Size, size distribution, zeta potential measurements and morphology of blank micelles and DOX-micelles Most of the drug-loaded mixed micelles obtaining methods usually employ organic solvents, which may be afterwards removed by conventional techniques [11]. In our case, DOX-micelles were prepared in a simple way, without the need of any organic solvents nor cryoprotectants for the lyophilization (Figure 1). In this way, there are certain features of our preparation method that make it a remarkable one, including a simple manufacturing process, controllable and ease of scaling-up. The Dh and size distribution of drug free and DOX-micelles before and after freezedrying process were characterized in terms of DLS at 25 °C (Table 1). On one hand, blank micelles exhibited a unimodal behavior with a Dh value of 13.2 ± 0.2 nm and a small PDI value (0.070 ± 0.015), in good agreement with previous results and consistent with a complete micellization process [12]. Interestingly, the DOXmicelles presented a unimodal distribution with slightly lower Dh values, when compared to their DOX-free counterparts, both before (10.7 ± 0.2 nm) and after

(10.4 ± 0.1 nm) the freeze-drying process. The non-variation in size is indicative of adequate stability during the lyophilization process. We have also observed a similar behavior after lyophilization of methotrexate loaded TPGS and NaDC micelles [18]. Also, the freeze-dried cake was red and easy to reconstitute in water (Figure 2). From a pharmaceutical point of view, this appears to be an advantage, as there was no need to employ any cryoprotectants, thus simplifying the formulation and reducing the costs of its preparation [19]. As shown in Table 1, zeta potential measurements resulted neutral for empty micelles as well as for DOX-micelles, both before and after the lyophilization process, with values ranging from -5.0 to -2.9 mV. Considering the presence of a non-ionic hydrophilic polymer (PEG) in the micellar corona, these neutral values were expected. Moreover, the DOX-micelles exhibited a narrow size distribution and a spherical morphology both before and after the freeze-drying process (Figure 2). For i.v. drug delivery, micelles should demonstrate their colloidal stability after their plasma dilution. In this sense, samples were diluted 1/50 in PBS pH 7.4 to simulate the dilution conditions of the i.v. administration employed in this study. In this case, the total volume of micelles was diluted in a total blood volume of approximately 1.5 mL [14]. On the one hand, DOX-micelles showed a bimodal pattern with a first size fraction of 12.2  ±  1.0 nm with lower intensity and a second size fraction of 184.4  ±  40.9 nm with higher intensity (Table 2). Besides, we have not observed any precipitation of the DOX-NaDC insoluble loaded complex when diluted with PBS. Furthermore, the drug concentration of the DOX-micelles was quantified after

the 1/50 dilution and approximately 98% of the drug concentration was maintained. On the other hand, Doxil® presented only one narrow size population of 79.4 ± 0.4 nm (Table 2). Interestingly, none of these formulations exhibited unimers upon dilution. Hence, their colloidal stability was demonstrated for a potential i.v. administration. Furthermore, the micellar size could be appropriate to promote passive drug targeting to solid tumors, an effect known as enhanced permeability and retention (EPR) [20-22]. 3.2 In vitro cytotoxicity In a previous work, our group has demonstrated an enhancement in the in vitro anticancer performance with the DOX-micelles in an ovarian cancer cell line (SKOV-3) and in a human triple negative breast cancer (TNBC) cell line (MDA-MB231), in comparison to the commercially available liposomal formulation known as Doxil® [12]. In this context, with the objective of comparing the in vitro cytotoxic performance of our DOX-micelles versus free DOX and Doxil®, 4T1 murine BC cells were exposed to different DOX concentrations of each formulation for 48 h. These BC cells were selected considering the clinical application of the drug, being the standard of care for TNBC. Furthermore, blank micelles were also evaluated. Encouraging results arose from this assay, as a significant decrement (p < 0.05) of the IC50 value was observed for the DOX-micelles (0.032 µg/mL) in comparison with Doxil® (0.053 µg/mL) (Table 3, Figure 3). The clinical relevance of these results lies in the lack of molecular therapeutic targets in 4T1 TNBC cells. Different from estrogen receptor expressing tumors, TNBC presents as an aggressive recurring disease, with a higher metastasizing frequency than other types of BC,

without hormonal or HER2 targeting therapies response and with less favorable prognosis than hormone receptor expressing tumors [23,24]. Besides, the mixed micellar formulation exhibited similar in vitro anticancer efficacy as the free drug, with no significant difference between their IC50 values (Table 3). Similar results were obtained in our previous work, where the DOX-micelles exhibited a significantly higher cytotoxicity than the liposomal preparation and a similar cytotoxic effect to the free drug in a human TNBC cell line (MDA-MB 231 cells) [12]. Also, this comparable in vitro cytotoxic effect between free DOX and micellar formulations employing different biomaterials has been also reported by several authors [25-28]. On the other hand, the lesser cytotoxic effect exhibited by Doxil® relative to the free drug and the DOX-micelles, may be likely due to the inadequate drug cellular uptake, as confirmed below in the in vitro internalization assay. The ineffective DOX release from the liposomes may play a certain role in these results, as previously suggested in other studies [29,30]. Hence, the results shown in 4T1 cancer cells reinforce the fact that our mixed micellar nanocarriers represent a potential alternative for cancer chemotherapy. Overall, the in vitro cytotoxicity results showed that the DOX-micelles could clearly improve the in vitro cytotoxic effect on a TNBC cell line (4T1), when compared to the commercially available liposomal formulation. 3.3 In vitro determination of apoptosis Flow cytometry was employed to detect apoptotic cells after staining with annexin V-FITC + PI in 4T1 BC cells left without treatment (control group) or treated either with free DOX, Doxil®, DOX-micelles or blank micelles (Figure 4).

The results showed that the DOX-micelles exhibited the highest rate of apoptotic cell death at its lowest concentration (0.05 µg/mL), in comparison to the control group and the rest of the treatments (p < 0.0001). Interestingly, the free DOX solution reached the same apoptotic effect only when its DOX concentration was 2fold higher than the DOX-micelles, indicating that half of the dose of the micellar formulation was necessary to obtain the same rate of apoptotic cell death as free DOX. For its part, it was observed that Doxil® at its highest concentration (0.1 µg/mL) exhibited less than half of the apoptotic effect than our DOX-micelles at their lowest concentration (0.05 µg/mL). Similar results were obtained when employing Pluronic F127-polyamidoamine dendrimer conjugates [31]. Moreover, no significant difference was observed between the control group and the blank micelles, suggesting that the apoptotic effect of the DOX-micelles would not be a consequence of the unloaded nanocarriers. 3.4 In vitro cellular uptake Considering that an antineoplastic agent must be internalized within tumor cells in order to achieve an acceptable anticancer effect, the in vitro cellular uptake of DOX from Doxil®, the free drug and the DOX-micelles have been assayed in 4T1 cancer cells over 6 h. There was a significant (p < 0.05) increase in the DOX intracellular content for the DOX-micelles, as compared to Doxil® and DOX solution at 2 and 6 h of incubation with 4T1 cells (Figure 5). Interestingly, as the incubation time increased, it was observed that the intracellular levels of DOX for the mixed micellar formulation strongly augmented, while this behavior was not observed for neither for the drug

solution nor Doxil®. These results suggest that the presence of TPGS could favor the increase in the intracellular DOX content of the micellar system, as previously reported by our group with other cancer cell lines (SKOV-3 and MDA-MB-231) [12]. Moreover, this improvement in the in vitro cellular uptake performance has been observed with other antineoplastic drugs using TPGS in different nanoparticulate systems [23,32-34]. To sum up, data confirms that DOX from our DOX-loaded mixed micellar system could be more efficiently in vitro internalized by 4T1 BC cells than from the rest of the formulations. 3.5 In vivo anticancer effect BC accounts for almost 23% of all cancers globally. Depending on the cancer type, stage and treatment, prognosis and survival rates vary greatly. Currently, there are four stages of BC, being stage IV a metastatic cancer with the least of favorable prognosis. In this sense, we chose 4T1 BC model, as 4T1 cells are highly malignant and is nowadays usually employed as an animal model for stage IV human BC [35]. In order to study the in vivo anticancer effect induced by the DOX-loaded micelles, we inoculated subcutaneously 1 x 105 4T1 cells / mouse in the right mammary fat pad of female BALB/c mice. Once the tumor reached approximately 100 mm3, the animals were randomly divided into 4 groups and were intravenously administered with saline solution, free DOX, Doxil® or DOX-micelles on days 0, 3 and 6 (Figure 6C). The tumor volume of each mouse was measured at 3, 6, 8, 11, 13, 15 and 18

days and analyzed in Figure 6A. At day 11, all of the DOX treatments presented significant slower tumor growth (p < 0.001) relative to the control group, evidencing the antitumoral effect of the drug. Furthermore, since day 15, the DOX-micelles and the free DOX groups exhibited significant slower tumor growth (p < 0.05) in comparison to Doxil®, suggesting that the micellar system presents higher in vivo anticancer effect than the commercially available liposomal formulation and equivalent potency to the free drug. One of the key concepts that can be considered is the bioavailability of the encapsulated drug. It has been stated that the mechanism of action of Doxil® relies on the liposomes being accumulated in the tumor and their content slowly released, thus the bioavailable drug would be able to produce its therapeutic effect. However, if a cytotoxic concentration of the released drug is not reached, there will not be any appreciable therapeutic response [36]. Moreover, it has been studied that nanocarriers ranging 5 nm (renal clearance threshold) to 100 nm with nearly neutral surface charge are considered as suitable for passive tumor accumulation, as a consequence of their ability of escaping tumor vasculature through endothelial fenestrae and their prolonged circulation time [37]. In a previous study, we have proved that DOX was in vitro released in a greater extent from our micellar system than from the commercially available liposomes [12]. This, together with the fact that the size of our DOXmicelles resulted optimal for passive tumor accumulation, may have played a certain role in the observed in vivo anticancer results. On day 18, the animals were sacrificed and the tumors were removed to observe their morphology and weight. Figure 6B shows the relative tumor weight ratio of

free DOX, Doxil® and DOX-micelles with respect to the average weight of the control group. The results reveal that the relative tumor weight of the DOX-micelles and free DOX are significantly (p < 0.05) lower than that of Doxil®. Moreover, when analyzing the tumor doubling times, it results clear that free DOX and DOXmicelles exhibit the highest values (5.6 ± 0.5 and 5.1 ± 0.2 days, respectively) with no significant difference between them, followed by Doxil® and the control group (Figure 6D). All these results imply that the administration of the DOX-micelles induced a significant tumor growth inhibition equivalent to that of DOX solution and higher than Doxil®, in accordance with the in vitro cytotoxicity results. Besides, histopathological

and immunohistochemical evaluations are also

important in assessing the anticancer potential of the DOX-loaded mixed micellar preparation (Figure 6E). Tumor necrosis occurs focally or in patches surrounded by a viable edge. The images are representative of the ones observed in the samples, in which increased necrosis is present in all treatments, comparing with the control group. Extensive necrotic areas predominated in free DOX and DOXmicelles treated tumors (Figure 6E, a-d). Additionally, PCNA (Figure 6E, e-h; Figure 6F) and TUNEL (Figure 6E, i-l; Figure 6F) results showed that DOXmicelles reduced PCNA proliferation marker expression (p < 0.05 versus control group) and significantly (p < 0.01) increased the rate of apoptotic cell death, in comparison to Doxil® and the control group. Overall, the results of the histopathological and immunohistochemical evaluations are congruent with those obtained in the in vitro cytotoxicity study and in the in vivo tumor growth inhibition assays, evidencing the superior antitumor efficacy of this novel nanotechnological

platform over the commercially available liposomal formulation in 4T1-tumor bearing BALB/c mice. 3.6 In vivo toxicity studies It is well known that one of the most serious side effects associated with the clinical use of DOX is the onset of cardiotoxicity. Moreover, it is worth stressing that this lethal adverse effect is cumulative and dose-dependent [3]. Therefore, the objective of reducing cardiotoxicity results of great clinical relevance. As mentioned before, in a previous study, we have already demonstrated that our mixed micellar formulation reduced the toxic effects of DOX in an in vivo zebrafish (Danio rerio) model, as this nanosystem presented less cardiotoxic and neurotoxic effects and less morphological alterations than the free drug [13]. In the present work,

cardiotoxicity

was

investigated

by

means

of

histopathological,

immunohistochemical and oxidative stress assays. In comparison to normal untreated hearts (Figure 7B, a), the cardiac tissue of DOX-treated animals displayed certain characteristics that evidenced serious damage, as severe cytoplasmatic and nuclear vacuolization, interstitial edema, karyolysis and vascular congestion (Figure 7B, b). These observations were also reported by several authors [38-40]. In contrast to the cardiac damage presented by the free drug, the hearts of the animals treated with Doxil® and DOX-micelles exhibited evident preservation of the cardiac structure with significantly reduced interstitial edema and vascular congestion (Figure 7B, c-d). Moreover, DOXtreated animals exhibited significantly increased expression of DNA damage

marker γH2AX than both Doxil® and DOX-micelles (Figure 7B, e-h). These results suggest that the mixed micelles could diminish the cardiac toxicity. Furthermore, we monitored the behavior of the animals as well as their body weight as a sign of systemic side effects of the drug (Figure 7A). However, no individual exhibited any abnormal behavior (data not shown) and the average body weight of any of the DOX-treated groups showed significant difference from the control group. Finally, TBARS levels were determined as a sign of oxidative stress in the cardiac tissue. However, no significant differences were recorded among the groups, indicating that probably this was not the main mechanism of cardiotoxicity (Figure 7C).

4. Conclusions In summary, our DOX-loaded mixed micelles were successfully prepared and lyophilized without the use of any cryoprotectants. This system could significantly enhance the intracellular drug concentration and its in vitro cytotoxicity in 4T1 BC cells. Above all, systemic administration of DOX-micelles to 4T1-tumor bearing BALB/c mice resulted in a significantly greater inhibition of breast tumor growth than Doxil® in this treatment protocol. Moreover, our DOX-loaded nanosystem presented significant less cardiotoxic effects than the free drug. Consequently, all these results demonstrate that our novel nanotechnological platform could successfully deliver DOX to the tumor with reduced cardiotoxicity, thus showing a promising clinical potential as an anticancer drug delivery carrier.

Declaration of interests None. Acknowledgements The authors are deeply grateful to the Universidad de Buenos Aires (Grants UBACyT 20020170100362BA and 20020170200370BA). MC is supported by doctoral scholarship of CONICET. MAM, EB, MZ, MBN, VAM and DAC are partially supported by CONICET, Argentina. The authors express their gratitude to Diego Giaquinta Romero and Mariano Portillo for technical support.

Figure and Table captions Figure 1. Representative scheme of the preparation method of the DOX-loaded mixed micellar formulation. Figure 2. TEM micrographs of DOX-micelles in distilled water and negatively stained with phosphotungstic acid solution (1% w/v) before and after freeze-drying process. Scale bars = 0.2 µm. Figure 3. Cell viability of 4T1 cells after 48 h of treatment with blank micelles, DOX-micelles, DOX solution and Doxil®. Results are expressed as mean ± S.D. (n = 3). Figure 4. Annexin-V staining assays were evaluated in 4T1 cells that were left untreated (control) or were treated with free DOX, Doxil®, DOX-micelles or blank micelles for 48 h (**** p < 0.0001 significant difference for DOX-micelles versus control, blank micelles, free DOX, and Doxil®). Figure 5. Time-dependent intracellular/cell DOX levels in 4T1 cell line for DOXmicelles in comparison with DOX solution and Doxil®. DOX content was normalized by protein concentrations of the cell lysates. Results are expressed as mean ± S.D. (n = 3). * The intracellular/cell DOX levels are significantly (p < 0.05) higher for DOXmicelles versus the drug solution. ** The intracellular/cell DOX levels are significantly (p < 0.05) higher for DOXmicelles versus Doxil®. Figure 6. In vivo anticancer effect of DOX-micelles on 4T1 tumor-bearing female BALB/c mice. (A) Tumor volume at 3, 6, 8, 11, 13, 15 and 18 days in 4T1--bearing BALB/c mice intravenously administered with free DOX, Doxil®, and DOX-micelles at days 0, 3 and 6 (4 mice per group; 2 mg/kg DOX equivalent per injection; *p < 0.05, **p < 0.01 versus Doxil®; ***p < 0.001 versus Control, One Way ANOVA / A posteriori Tukey). (B) Relative tumor weight ratio of free DOX, Doxil® and DOXmicelles compared to the average weight of the control group (*p < 0.05 versus

Doxil®; *p < 0.05 versus Control, One Way ANOVA / A posteriori Tukey). (C) Schematic representation of the treatment protocol. A week before the first injection, mice were inoculated with approximately 1 x 105 4T1 cells. When the tumors reached approximately 100 mm3, the first i.v. administration was carried out and was followed by 2 other injections on days 3 and 6, respectively. The sacrifice of the animals was performed on day 18. (D) Median tumor doubling time of each group is shown numerically (*p < 0.05 versus Doxil®; *p < 0.05 versus Control, One Way ANOVA / A posteriori Tukey). (E) Histopathological and immunohistochemical analyses of tumor tissues. (a-d) Representative H&E-stained sections showing that increased necrosis is present in all treatments, comparing with the control group. Extensive necrotic areas predominated in free DOX and DOX-micelles treated tumors. (e-h) TUNEL and (i-l) PCNA in paraffin-embedded tumor tissues. x400 Original magnification. (F) The number of tumor cells and the percentage of PCNA (*p < 0.05 versus control) and TUNEL (**p < 0.01 versus Control; **p < 0.01 versus Doxil®) positive stained cells were quantified by counting 10 random fields. Figure 7. DOX-micelles decrease DOX-induced cardiotoxicity in BALB/c mice. (A) Average body weight analysis of the different groups throughout the study. All the mice maintained or even increased their body weight, proving their good health conditions all over the experiment. (B) (a-d) Representative H&E stained specimens and (e-h) immunohistochemical images of γH2Ax sections are shown. (a) Normal histological appearance of an untreated heart. (b) Heart of DOX-treated animals displaying severe cytoplasmatic and nuclear vacuolization, interstitial edema, karyolysis and vascular congestion. (c-d) Heart of Doxil® and DOXmicelles treated animals showing evident preservation of the cardiac structure with significantly

reduced

interstitial

edema

and

vascular

congestion.

(e)

Immunohistochemical image of an untreated γH2Ax section. (f-h) Red arrows indicate DNA damage in the cardiac cells. Evidently, DOX induces more DNA damage than Doxil® and DOX-micelles, while there are no differences among the latter. (C) DOX-micelles effects on TBARS levels of female BALB/c mice. TBARS levels were determined in mice heart. Data are expressed as nmol/mg of tissue (4 mice per group, no significant difference between groups).

Table 1. Micellar size, size distribution (PDI) and zeta potential of the blank micelles and DOX-micelles at 25 °C, before and after lyophilization process. Table 2. Size and size distribution of DOX-micelles and Doxil® diluted 1/50 in PBS pH 7.4 to simulate the dilution process after i.v. administration, measured by DLS at 37 °C. Table 3. IC50 (mean ± S.D.) values in 4T1 cells after 48 h treatment by Doxil®, DOX solution, blank micelles and DOX-micelles.

Table 1. Micellar size, size distribution (PDI) and zeta potential of the blank micelles and DOX-micelles at 25 °C, before and after lyophilization process.

Sample Blankmicelles DOXmicelles

Before lyophilization Zeta Size PDI potential (nm) (mV) 13.2 0.070 -5.0 (1.5) (0.2) (0.015) 10.7 0.239 -2.9 (0.6) (0.2) (0.010)

After lyophilization Zeta Size PDI potential (nm) (mV) -

-

-

10.4 (0.1)

0.221 (0.018)

-4.1 (0.8)

Table 2. Size and size distribution of DOX-micelles and Doxil® diluted 1/50 in PBS pH 7.4 to simulate the dilution process after i.v. administration, measured by DLS at 37 °C.

Nanosystems DOX-micelles Doxil®

Peak 1 Peak 2 PDI (±S.D.) Dh (nm) (±S.D.) % Dh (nm) (±S.D.) % 12.2 (1.0) 41.7 184.4 (40.9) 58.3 0.313 (0.050) 79.4 (0.4) 100 0.079 (0.014)

Table 3. IC50 (mean ± S.D.) values in 4T1 cells after 48 h treatment by Doxil®, DOX solution, blank micelles and DOX-micelles. IC50 (µg/mL)

Cell line 4T1

DOX solution

Doxil®

DOX-micelles

0.028 ± 0.004a

0.053 ± 0.012b,c

0.032 ± 0.004a

Blank micelles >1

Note: Multiple comparisons were performed using one-way ANOVA (n = 6 experiments). a significant difference compared to Doxil® (p < 0.05) b significant difference compared to DOX solution (p < 0.05) c significant difference compared to DOX-micelles (p < 0.05)

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Conflicts of interest The authors declare no conflict of interest.