In vivo antitumour properties of tribenzyltin carboxylates in a 4T1 murine metastatic mammary tumour model: Enhanced efficacy by PLGA nanoparticles

Journal Pre-proof

In vivo antitumour properties of tribenzyltin carboxylates in a 4T1 murine metastatic mammary tumour model: Enhanced efficacy by PLGA nanoparticles

Theebaa Anasamy ConceptualizationMethodologyInvestigationWriting- Original draft preparation , Chin Fei Chee ConceptualizationMethodologyResourcesWriting- Reviewing and Editing , Lik Voon Kiew ConceptualizationResourcesWriting- Reviewing and Editing , Lip Yong Chung ConceptualizationWriting- Reviewing and EditingSupervisionProject AdministrationFunding Acquis PII: DOI: Reference:

S0928-0987(19)30413-0 https://doi.org/10.1016/j.ejps.2019.105140 PHASCI 105140

To appear in:

European Journal of Pharmaceutical Sciences

Received date: Revised date: Accepted date:

29 August 2019 11 October 2019 4 November 2019

Please cite this article as: Theebaa Anasamy ConceptualizationMethodologyInvestigationWriting- Original draft prep Chin Fei Chee ConceptualizationMethodologyResourcesWriting- Reviewing and Editing , Lik Voon Kiew ConceptualizationResourcesWriting- Reviewing and Editing , Lip Yong Chung ConceptualizationWr In vivo antitumour properties of tribenzyltin carboxylates in a 4T1 murine metastatic mammary tumour model: Enhanced efficacy by PLGA nanoparticles, European Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.ejps.2019.105140

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.

Page 1 of 35

In vivo antitumour properties of tribenzyltin carboxylates in a 4T1 murine metastatic mammary tumour model: Enhanced efficacy by PLGA nanoparticles Theebaa Anasamya, Chin Fei Cheeb, Lik Voon Kiewc and Lip Yong Chunga,* a

Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala

Lumpur, Malaysia b

Nanotechnology & Catalysis Research Centre, University of Malaya, 50603 Kuala Lumpur,

Malaysia c

Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala

Lumpur, Malaysia *Corresponding author. Email address: [email protected], [email protected] (L. Y. Chung) Address: Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Phone number: +603 7967 7834 Fax: +603 7967 4964 ABSTRACT This study reports the in vivo performance of two tribenzyltin carboxylate complexes, tri(4fluorobenzyl)tin[(N,N-diisopropylcarbamothioyl)sulfanyl]acetate

(C1)

and

tribenzyltin

isonicotinate (C9), in their native form as well as in a poly(lactic-co-glycolic acid) (PLGA)based nanoformulation, to assess their potential to be translated into clinically useful agents. In a 4T1 murine metastatic mammary tumour model, single intravenous administration of C1 (2.7 mg/kg) and C9 (2.1 mg/kg; 2.1 mg/kg C9 is equivalent to 2.7 mg/kg C1) induced greater tumour growth delay than cisplatin and doxorubicin at equivalent doses, while a double-dose regimen demonstrated a much greater tumour growth delay than the single-dose treated groups. To improve the efficacy of the complexes in vivo, C1 and C9 were further integrated into PLGA nanoparticles to yield nanosized PLGA-C1 (183.7 ± 0.8 nm) and

Page 2 of 35 PLGA-C9 (163.2 ± 1.2 nm), respectively. Single intravenous administration of PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGA-C9 (2.1 mg C9 equivalent/kg) induced greater tumour growth delay (33% reduction in the area under curve compared to that of free C1 and C9). Multiple-dose administration of PLGA-C1 (5.4 mg C1 equivalent/kg) and PLGA-C9 (4.2 mg C9 equivalent/kg) induced tumour growth suppression at the end of the study (21.7 and 34.6% reduction relative to the size on day 1 for the double-dose regimen; 73.5 and 79.0% reduction relative to the size on day 1 for the triple-dose regimen, respectively). Such tumour growth suppression was not observed in mice receiving multiple-dose regimens of free C1 and C9. Histopathological analysis revealed that metastasis to the lung and liver was inhibited in mice receiving PLGA-C1 and PLGA-C9. The current study has demonstrated the improved in vivo antitumour efficacies of C1 and C9 compared with conventional chemotherapy drugs and the enhancement of the efficacies of these agents via a robust PLGA-based nanoformulation and multiple-drug administration approach. Keywords: Triorganotin carboxylates

Poly(lactic-co-glycolic acid) Metastatic breast cancer 4T1 murine mammary tumour model Enhanced permeability and retention effect 1.

INTRODUCTION

Organotin(IV) derivatives have been reported to exhibit good anticancer activities in vitro (e.g., against lymphoma, cervical, breast and colon cancer cell lines) and in vivo (e.g., in sarcoma-bearing female Wistar rats for the triphenyltin complex and in BDF1 mice bearing

Page 3 of 35 melanoma tumours for the tributyltin complex) (Alama et al., 2009; Navakoski de Oliveira et al., 2014; Sirajuddin and Ali, 2016; Varela-Ramirez et al., 2011; Verginadis et al., 2011). While earlier investigations mainly focused on the anticancer properties of triorganotin species such as tributyltin and triphenyltin derivatives, our previous investigation revealed that two tribenzyltin derivatives, i.e., tri(4-fluorobenzyl)tin[(N,N-diisopropylcarbamothioyl) sulfanyl]acetate (C1) and tribenzyltin isonicotinate (C9), possess better activity than their respective triphenyltin analogues when tested using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay. These complexes exhibited prominent anticancer activity with higher selectivity for breast cancer cells (approximately 20- to 70-fold) compared to normal breast epithelial cells than cisplatin and doxorubicin (2- and 5-fold, respectively) (Anasamy et al., 2017). Moreover, these complexes reduced the migration and invasion (up to 90%) of the highly aggressive breast cancer cells MDA-MB231 and 4T1 when compared to the control group (Anasamy et al., 2017). Further investigations on the mechanism of cell death induced by C1 and C9 revealed that these complexes caused cellular morphological changes related to apoptosis, phosphatidylserine externalisation, changes in the cell cycle distribution and activation of caspases-3/7, -8 and -9 indicating the involvement of both death receptor and mitochondrial pathway of apoptosis (Anasamy et al., 2017). Nevertheless, the in vivo toxicity and antitumour efficacy of C1 and C9 or similar compounds of the tribenzyltin class have yet to be reported. Poor drug solubility remains a major drawback of triorganotin complexes, which potentially limits their efficacy in an in vivo setting. For instance, the tribenzyltin complexes investigated in this study were found to possess high lipophilicity (log P > 4) and low aqueous solubility (< 70 µM) (Supplementary Material (Fig. S1). This could result in

Page 4 of 35 unpredictable in vivo outcomes if administered directly (Lu and Park, 2013). The formulation of lipophilic anticancer agents, e.g., paclitaxel and docetaxel, with FDAapproved excipients, such as polyethylated castor oil/ethanol (e.g., Kolliphor EL) or polysorbate 80/ethanol has been commonly practiced in clinical settings to improve the aqueous solubility and stability of the anticancer agents (Binder, 2013). However, the use of these excipients may risk the induction of adverse effects, including hypersensitivity reactions, neutropenia and sensory neuropathy in patients (Binder, 2013; ten Tije et al., 2003; Weiss et al., 1990). Formulating anti-cancer drugs using nanodelivery systems may potentially overcome these obstacles (Acharya and Sahoo, 2011; Kumari et al., 2010). Thus, C1 and C9 were further integrated into biocompatible poly(lactic-co-glycolic acid) (PLGA) nanoparticles to improve their blood solubility, reduce random tissue distribution, prolong blood circulation time of the drugs and passively increase the accumulation of drugs in tumour tissues via enhanced permeability and retention effects (Danhier et al., 2012; Gaillard et al., 2014; Kumari et al., 2010; Öztürk-Atar et al., 2017). Accordingly, PLGA nanoparticles containing tribenzyltin carboxylates were prepared using the nanoprecipitation method and examined for their physicochemical properties, in vitro release profiles, in vivo toxicity profiles, antitumour efficacies and antimetastatic properties. Herein, we report the in vivo toxicity and antitumour efficacy of C1 and C9 (i) as free agents in single/multiple dosing regimens and (ii) as PLGA-nanoformulated agents in single/multiple dosing regimens against a 4T1 metastatic murine mammary cancer model using cisplatin and doxorubicin for comparison. 2.

MATERIALS AND METHODS

2.1

Materials and reagents

Page 5 of 35 Acetic acid, hydrochloric acid (37%), sodium hydroxide, dimethyl sulfoxide and acetonitrile were supplied by Merck (Darmstadt, Germany). Poly(D,L-lactide-co-glycolide) (50:50, MW 38,000 – 54,000) and poly(vinyl alcohol) (PVA) (MW 30,000 – 70,000) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Phosphate-buffered saline (PBS) and RPMI 1640 cell culture media were obtained from Biowest (Missouri, USA). Sodium lauryl sulfate (SLS) was obtained from Amresco (Ohio, USA). Deionized water with a resistivity of 18.2 ΩS cm-1 obtained from the Barnstead Nanopure® Diamond™ RO system (Missouri, USA) was used throughout the study. Other materials used for specific experiments are described in their respective methodology sections. 2.2 The

Tribenzyltin carboxylates tribenzyltin

carboxylates,

tri(4-fluorobenzyl)tin

[(N,N-

diisopropylcarbamothioyl)sulfanyl]acetate (C1) and tribenzyltin isonicotinate (C9), were synthesized and characterized as previously reported (Anasamy et al., 2017). The chemical structures of C1 and C9 are shown in Fig. 1. 2.3

In vivo studies

Female wild-type BALB/c mice 7

8 weeks old weighing between 18 – 20 g were

purchased from the Animal Experimental Unit (AEU), Faculty of Medicine, University of Malaya, Malaysia for in vivo studies. The mice were housed in a satellite animal facility at the Department of Pharmacology, Faculty of Medicine, University of Malaya under controlled temperature and humidity, a 12 h light-dark cycle environment, and with ad libitum access to food and water. All applicable institutional guidelines for the care and use of animals were followed (Faculty of Medicine Institutional Animal Care and Use Committee, University of Malaya. Ethics Reference no. 2016-190503/PHARM/R/TA).

Page 6 of 35 2.4

In vivo toxicity study of free C1 and C9

The in vivo toxicity profiles of cisplatin (positive control), free C1 and C9 were evaluated following intravenous administration of the samples to healthy female BALB/c mice via the tail vein. The toxicity was tested at concentrations of 15, 12.5 and 10 mg/kg for C1; 7.2, 6 and 4.8 mg/kg for C9 and 10, 8 and 6 mg/kg for cisplatin. All compounds were dissolved in a cocktail of 5% ethanol and 5% Cremophor EL and diluted with saline to give 0.2 ml. Each mouse was administered with 0.2 ml of the samples. Potential toxicity was observed for 3 weeks based on symptoms such as inactivity, ruffled fur, behavioural changes, and loss of body weight (Koudelka et al., 2010). 2.5

In vivo antitumour efficacy of free C1 and C9 in 4T1 tumour-bearing mice

4T1 tumour transplantation was carried out by subcutaneous injection of murine 4T1 breast cancer cells (5 × 105 cells) in 0.1 ml of RPMI medium into the mammary pad of the mice. When the tumour reached an average volume of approximately 100 mm3, the mice were intravenously injected via the tail vein with the test samples (Wehbe et al., 2018; Yokoyama et al., 1991). Free C1 and C9 as well as cisplatin were dissolved in a cocktail of ethanol and Cremophor EL (1:1). The mixtures were then diluted in normal saline to give a volume of 0.2 ml per injection containing 5% ethanol and 5% Cremophor EL. The mice were randomly divided into 8 groups (n = 7) with treatments as follows: (a) single dose of free C1 (2.7 mg/kg), (b) single dose of free C9 (2.1 mg/kg; 2.1 mg/kg C9 is equivalent to 2.7 mg/kg C1), (c) repeated doses of free C1 (2.7 mg/kg, days 1 and 7)††, (d) repeated doses of free C9 (2.1 mg/kg, days 1 and 7)††, (e) single dose of cisplatin (1.2 mg/kg; 1.2 mg/kg cisplatin is equivalent to 2.7 mg/kg C1 and 2.1 mg/kg C9), (f) single dose of doxorubicin (2.2 mg/kg; 2.2 mg/kg doxorubicin is equivalent to 2.7 mg/kg C1 and 2.1 mg/kg C9), (g) saline and (h)

Page 7 of 35 saline mixed with a cocktail of 5% ethanol and 5% Cremophor EL. ††Indicates a doubledose regimen administered on days 1 and 7. Free C1 (2.7 mg/kg), free C9 (2.1 mg/kg), cisplatin (1.2 mg/kg) and doxorubicin (2.2 mg/kg) are equivalent to 4 µmol/kg. Tumour volumes were measured with a calliper (TESA Technology, Renens, Switzerland) and calculated using a previously described formula (Voon et al., 2016). Next, the area under the curve (AUC) was calculated for each group using GraphPad Prism version 8.1.1 (GraphPad, San Diego, CA, USA) (Duan et al., 2012). 2.6

Synthesis and characterisation of PLGA-C1 and PLGA-C9 nanoparticles

2.6.1 Evaluation of surfactant concentration as a formulation variable The effect of the surfactant concentration on the physicochemical parameters of blank PLGA nanoparticles was determined by using different concentrations of PVA, i.e., 0.5, 1.0, 1.5 and 2.0% (w/v) in the aqueous phase. The PVA concentration that gives the most desirable physicochemical characteristics of blank PLGA was chosen for the preparation of C1- and C9-loaded PLGA nanoparticles. 2.6.2 Preparation of C1- and C9-loaded PLGA nanoparticles PLGA nanoparticles loaded with C1 or C9 were prepared following a previously reported nanoprecipitation technique with minor modifications (Yallapu et al., 2010). PLGA was dissolved in acetonitrile at a concentration of 30 mg/ml and mixed with 10 mg of C1 or C9. The mixture was stirred for 5 min and added dropwise to 25 ml of aqueous phase containing PVA as a surfactant at room temperature under magnetic stirring. The reaction mixture was stirred overnight to remove traces of acetonitrile. The supernatant containing PLGA-C1 or PLGA-C9 was recovered by ultracentrifugation at 102,000 × g for 25 min at 4 °C using a 70 Ti Rotor (Beckman Coulter, California, USA). The pellet was resuspended in water and kept

Page 8 of 35 at -80 °C overnight before being freeze-dried for 36 h using a freeze-dryer (Labconco, Missouri, USA). The resulting nanoparticles were stored in a desiccator at 4 °C until further use. Similarly, blank PLGA nanoparticles were prepared by dissolving PLGA in acetonitrile without C1 or C9, following the same procedure. 2.6.3 Determination of the process yield, loading and entrapment efficiency Freeze-dried PLGA-C1 and PLGA-C9 nanoparticles were dissolved in acetonitrile for the extraction of C1 and C9 to estimate the loading and entrapment efficiency. The samples were dissolved in acetonitrile and placed on a shaker (Boeco, Hamburg, Germany) at 100 rpm for 24 h to separate C1 and C9 from the nanoparticles. The samples were then centrifuged at 9,300 × g for 10 min, and the supernatant was subjected to dilution prior to quantification using a UV-Vis spectrophotometer (Perkin-Elmer, Massachusetts, USA) at 250 nm for C1 and 255 nm for C9. The concentration of the complexes was calculated using a standard plot of C1 and C9 (0 – 20 µg/ml). The process yield (%), loading (%, w/w) and entrapment efficiency (%) were calculated using previously described methods (Voon et al., 2016; Yallapu et al., 2010). 2.6.4 Particle size and zeta potential The size of the nanoparticles was determined by using a Malvern Nanoseries Zetasizer (Malvern, Worcestershire, UK) based on the dynamic light scattering principle (Baalousha et al., 2012; Murdock et al., 2008). To measure the particle size, 50 µl of a 1 mg/ml nanoparticle suspension was added to 3 ml of deionized water and sonicated for 30 s prior to measurement. To examine the nanoparticle stability, the prepared nanoparticles were monitored by using a Malvern Nanoseries Zetasizer over 30 days to detect cluster formation

Page 9 of 35 at ambient temperature and 4 °C. Zeta potential was measured based on the principle of electrophoretic mobility under an electric field (Deryabin et al., 2015). 2.6.5 Particle morphology The nanoparticles were characterized for their morphology using a scanning electron microscope (Hitachi FESEM SU8000, Tokyo, Japan) operating at an accelerating scanning voltage of 0.5 – 30 kV. Nanoparticles were air-dried in a desiccator and coated with gold prior to examination by scanning electron microscopy. 2.7

In vivo toxicity study of PLGA-C1 and PLGA-C9 nanoparticles

The in vivo toxicity profile of PLGA-C1 and PLGA-C9 nanoparticles as well as blank PLGA nanoparticles was determined following intravenous administration of the samples to healthy female BALB/c mice via the tail vein. 2.8

In vivo antitumour efficacy studies of PLGA-C1 and PLGA-C9 nanoparticles in

4T1 tumour-bearing mice PLGA-C1 and PLGA-C9 nanoparticles were prepared in normal saline to a volume of 0.2 ml per injection. The mice were randomly divided into 9 groups (n = 7) with treatments as follows: (a) single dose of PLGA-C1 NP, containing 2.7 mg equivalent C1/kg; (b) single dose of PLGA-C1 NP, containing 5.4 mg C1 equivalent/kg; (c) repeated doses of PLGA-C1 NP, containing 5.4 mg C1 equivalent/kg (days 1 and 7)††; (d) repeated doses of PLGA-C1 NP, containing 5.4 mg C1 equivalent/kg (days 1, 5 and 9)†††; (e) single dose of PLGA-C9 NP, containing 2.1 mg C9 equivalent/kg; (f) single dose of PLGA-C9 NP, containing 4.2 mg C9 equivalent/kg; (g) repeated doses of PLGA-C9 NP, containing 4.2 mg C9 equivalent/kg (days 1 and 7)††; (h) repeated doses of PLGA-C9 NP, containing 4.2 mg C9 equivalent/kg (days 1, 5 and 9)†††; and (i) blank PLGA nanoparticles. ††Indicates a double dose,

Page 10 of 35 administered on days 1 and 7, while ††† indicates a triple dose, administered on days 1, 5 and 9. PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGA-C9 (2.1 mg C9 equivalent/kg) are equivalent to 4 µmol/kg C1 and C9, respectively. While PLGA-C1 (5.4 mg C1 equivalent/kg) and PLGA-C9 (4.2 mg C9 equivalent/kg) are equivalent to 8 µmol/kg C1 and C9, respectively. Tumour volumes were measured with a calliper (TESA Technology) and calculated using a previously described formula (Voon et al., 2016). Next, the AUC was calculated for each group using GraphPad Prism version 8.1.1 (Duan et al., 2012). 2.9

Histopathological evaluation

The major organs harvested from mice after 15 days of treatment were preserved in 10% neutral-buffered formalin solution prior to the staining procedure. The tissues were then processed in paraffin, stained with haematoxylin and eosin and viewed under a light microscope with a 40× objective (Nikon Eclipse E200 attached to a Nikon Digital Sight camera, Tokyo, Japan). 2.10

Statistical analysis

All values are expressed as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 8.1.1 (GraphPad Software Inc., San Diego, California) using one-way ANOVA and two-way ANOVA paired with Tukey’s post hoc test for repeated measures with a significance level set at p < 0.05. 3.

RESULTS AND DISCUSSION

3.1

In vivo toxicity and maximum tolerable dose of cisplatin, C1 and C9

The toxicity profiles of the studied complexes are crucial in determining the dose of a formulation and can be a predictor of clinical success. The in vivo toxicity profiles of

Page 11 of 35 cisplatin, C1 and C9 were evaluated following intravenous administration of these samples to mice via the tail vein at different doses (Fig. 2). Cisplatin was well tolerated at doses of 6 mg/kg and 4.8 mg/kg. However, the body weight of mice administered 7.2 mg/kg cisplatin dropped below the acceptable weight loss, and all the mice died on day 7 after drug administration. Reduced movement and ruffled fur symptoms were also observed. These results are in agreement with the toxicity report of cisplatin from other studies (ranging from 4 to 7 mg/kg for a single dose administration and 3 to 5.4 mg/kg for multiple dose administration) (Kai et al., 2015; Li et al., 2015; Mattheolabakis et al., 2009; Xiong et al., 2012). C1 and C9 were well tolerated at doses of 12.5 mg/kg and 8 mg/kg, respectively. 3.2

In vivo antitumour efficacy of free C1 and C9

Free C1 and C9 showed better therapeutic efficacy in 4T1 tumour-bearing mice compared to those treated with cisplatin and doxorubicin (Fig. 3). The area under curve (AUC) for both the C1 (2.7 mg/kg, single dose) and C9 (2.1 mg/kg, single dose) was significantly reduced by 50.5% and 50.8% (p < 0.0001, one-way ANOVA, Tukey’s post hoc test), respectively, when compared to the saline group and 49.8% and 50.1% (p < 0.0001, one-way ANOVA, Tukey’s post hoc test), respectively, when compared to the vehicle treated group. The AUC for the free C1 (2.7 mg/kg, single dose) treated group was reduced by approximately 17.5% and 28.9% more than the cisplatin- and doxorubicin-treated groups, respectively. Moreover, in the free C9 (2.1 mg/kg, single dose) treated group, the AUC was reduced by approximately 18.2% and 29.4% more than the cisplatin and doxorubicin treated groups, respectively. Small molecules are easily eliminated from the body via renal excretion (Maeda et al., 2009). To compensate for such loss, a multiple dosing regimen was adopted in the current

Page 12 of 35 study (Arnold et al., 2005). Mice treated with multiple doses of C1 and C9 showed a better tumour growth delay than those treated with a single dose of C1 and C9. Multiple dose administration of C1 (2.7 mg/kg, days 1 and 7) and C9 (2.1 mg/kg, days 1 and 7) over 15 days reduced the AUC by 33.8% and 25.9%, respectively, compared to the corresponding single-dose treatment. When compared with the saline control group, the AUC of the twodose C1 and C9 treated groups was significantly reduced by 67.3% and 63.2% (p < 0.0001, one-way ANOVA, Tukey’s post hoc test), respectively. Overall, the in vivo experiments using free C1 (2.7 mg/kg) and C9 (2.1 mg/kg) at single and multiple dose(s) demonstrated a dose- and regimen-dependent delay in tumour growth, and the effect was higher compared to that of cisplatin and doxorubicin at equivalent concentrations. Previously, several studies reported the in vivo anticancer properties of organotin complexes. For instance, sarcoma-bearing female Wistar rats treated with 4 doses of a triphenyltin complex with a 2-mercaptonicotinic acid ligand (2.1 mg/kg) did not show tumour growth delay or regression, although the mean survival time increased by 47% compared to the control group (Verginadis et al., 2011). A five-dose treatment protocol of bimetallic (tin (Sn) and silver (Ag)) supramolecular coordination polymers (3.5 mg/kg) treated on breast tumour-bearing female Wistar rats only caused tumour growth delay up to 44.8% after 15 days of treatment (Etaiw et al., 2011), whereas C1 and C9 reduced the AUC up to 51% after a single-dose treatment of 2.7 and 2.1 mg/kg for C1 and C9, respectively when compared to the control group. Moreover, the two-dose treatment of C1 and C9 reduced the AUC by approximately 67.3% and 63.2%, respectively, after 15 days of treatment. In a murine colon solid tumour model, an oxaliplatin-based platinum complex did not show any antitumour effects in a four-dose treatment regimen, while a platinum

Page 13 of 35 derivative with a 1,10-phenanthroline ligand only showed a similar in vivo tumour growth delay to that of cisplatin in a murine Lewis lung solid tumour model (Göschl et al., 2017; Harper et al., 2017). In contrast, C1 and C9 showed better tumour growth delay than cisplatin, which were 17.8% and 18.2% more, respectively, based on the AUC. Despite being able to delay tumour growth, complete tumour suppression was not attained by the free tribenzyltin complexes. High lipophilicity (log P > 4), low aqueous solubility (< 70 µM) and the small molecular size of the tribenzyltin complexes, which may lead to body fat deposition (Chen et al., 2007), random distribution (Park et al., 2010) and unsatisfactory tumour availability in spite of the intravenous administration, may be the main causes. To explore the possibility of a further reduction of in vivo toxicity and to improve the antitumour efficacy of C1 and C9, C1 and C9 were integrated into a biocompatible PLGAbased nanodrug delivery system. 3.3

PLGA-NP, PLGA-C1 and PLGA-C9: Summary of characteristics

The basic physicochemical characteristics of the synthesised PLGA-C1 and PLGA-C9 nanoparticles, as well as blank PLGA carrier nanoparticles (PLGA-NP), are shown in Table 1. Further characterisation data of PLGA-C1 and PLGA-C9 (scanning electron microscopy, 30 days stability study, in vitro aqueous/plasma release kinetics) are described in Supplementary Material (Table S1

S2,

).

Page 14 of 35 3.4

In vivo toxicity and maximum tolerable dose of PLGA-NP, PLGA-C1 and

PLGA-C9 PLGA-C1 and PLGA-C9 were well tolerated at higher doses than free C1 and C9, at doses equivalent to the free drug doses of 17.5 mg/kg and 12 mg/kg, respectively (Fig. 4(a) and Fig. 4(b)). This was in line with previous studies that reported that drugs such as docetaxel, cisplatin and betulinic acid encapsulated in PLGA nanoparticles have lower toxicity profiles than the free drug, as systemic exposure is reduced in an encapsulated form (Bowerman et al., 2017; Mattheolabakis et al., 2009; Saneja et al., 2017). Mice which were administered PLGA-NP (a blank PLGA nanoparticle with equivalent concentrations of the highest PLGAC1 and PLGA-C9 doses) remained healthy throughout the study period, indicating that the biodegradable carrier has no or minimal side effects. In addition, PLGA-C1 and PLGA-C9 were well tolerated at higher doses than their corresponding free drugs, reflecting the protection offered by the nanodelivery system against the nonspecific toxicity of the small molecular therapeutics. 3.5

In vivo antitumour efficacy of PLGA-C1 and PLGA-C9 nanoparticles

Single-dose administration of PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGA-C9 (2.1 mg C9 equivalent/kg) nanoparticles showed enhanced tumour growth delay compared with the free C1 and C9 treated groups at equivalent doses, respectively (Fig. 5). The AUC was reduced by 33% in the treatment group administered PLGA-C1 (2.7 mg C1 equivalent/kg) compared with the free C1 group at the equivalent dose (p < 0.05, one-way ANOVA, Tukey’s post hoc test), while in the PLGA-C9 (2.1 mg C9 equivalent/kg) treatment group, the AUC was reduced by 23.5% compared with the free C9 (2.1 mg/kg) group (p > 0.05, one-way ANOVA, Tukey’s post hoc test). In comparison to the carrier control group,

Page 15 of 35 PLGA-C1 (single dose, 2.7 mg C1 equivalent/kg) and PLGA-C9 (single dose, 2.1 mg C9 equivalent/kg) reduced the AUC significantly by 69.5% and 63.1% (p < 0.0001, one-way ANOVA, Tukey’s post hoc test), respectively. Single-dose administration of PLGA-C1 and PLGA-C9 at a higher dose (5.4 mg C1 or 4.2 mg/kg C9) caused a temporary suppression in tumour volume by 36.7% and 25.3%, respectively, after the treatment on day 1. Subsequently, a tumouristatic effect was observed until day 4, whereby the tumour volume remained unchanged, and after day 4, the tumour volume gradually increased until day 15 (2× increase in volume relative to the volume on day 1). To further improve the therapeutic efficacy, the multiple-dose administration of PLGA-C1 (Fig. 5(a)) and PLGA-C9 nanoparticles (Fig. 5(b)) was carried out. A doubledose administration of PLGA-C1 (5.4 mg C1 equivalent/kg, days 1 and 7) and PLGA-C9 (4.2 mg C9 equivalent/kg, days 1 and 7) successfully suppressed the tumour volume by approximately 21.7% and 34.6% on day 15, respectively, compared to the initial tumour volume on day 1. Meanwhile, triple-dose administration of PLGA-C1 (5.4 mg C1 equivalent/kg, days 1, 5 and 9) and PLGA-C9 (4.2 mg C9 equivalent/kg, days 1, 5 and 9) further suppressed the tumour volume by 73.5% and 79.0% on day 15, respectively, compared to the initial tumour volume on day 1. The increased efficacy of PLGA-C1 and PLGA-C9 could be ascribed to the increased circulatory potential, which consequently led to the higher passive accumulation of nanocarrier in the tumour tissues through the enhanced permeability and retention effect (Acharya and Sahoo, 2011). A previous study reported the in vivo antitumour activity of the triphenyltin hexanol (Sn) complex encapsulated in mesoporous silica nanoparticles (SBA-15p) (Bulatovic et al., 2014). Triphenyltin hexanolcontaining

mesoporous

silica

nanoparticles

(SBA‐ 15pSn)

were

administered

Page 16 of 35 intraperitoneally to C57BL/6 mice bearing B16 melanoma at 9 mg/kg (three times a week for 28 days), and the tumour volume was determined at the end of the study. In mice treated with the triphenyltin compound alone, tumour growth delay was observed, while the administration of SBA‐ 15pSn nanoparticles almost completely suppressed the tumour volume after a 12-dose regimen of 9 mg/kg over 28 days. In contrast, PLGA-C1 (5.4 mg C1 equivalent/kg) and PLGA-C9 (4.2 mg C9 equivalent/kg) nanoparticles almost completely suppress the tumour volume after a triple-dose administration (over 15 days) at a much lower-dose administration (4.2 to 5.4 mg/kg). This indicates that PLGA nanoparticles could be explored as an alternative for the delivery of organotin complexes. Moreover, a previous study using a photosensitizer encapsulated in PLGA nanoparticles (PLGA-I2BODIPY) prepared via similar nanoprecipitation method

revealed that the PLGA-I2BODIPY

nanoparticles exhibited significant and prolonged accumulation in the tumour tissues (Voon et al., 2016). The fluorescence intensity of tumour in mice treated with PLGA-I2BODIPY nanoparticles was approximately 4.5 times higher than the intensities of those treated with free photosensitizer, at 1-hour post-administration, indicating preferential accumulation of the nanoparticles in the tumour tissues, most likely via the exploitation of enhanced permeability and retention effect (Voon et al., 2016). PLGA-I2BODIPY nanoparticles also have similar physicochemical properties as PLGA-tribenzyltin complexes, such as size (~ < 180 nm) and the polydispersity index (PDI < 0.08). The cellular uptake study using 4T1 breast cancer cell lines also revealed increased cell uptake of the PLGA-I2BODIPY nanoparticles (1.5 times higher) compared to that of the free photosensitizer (Voon et al., 2016). These data suggest that the biodistribution of PLGA-tribenzyltin is likely to be similar to that of PLGA-I2BODIPY.

Page 17 of 35 3.6

Histopathological analysis

Major organ (kidney, liver and lung) histology was performed for various mice groups, i.e., healthy mice control group; saline-treated 4T1 tumour-bearing mice control group; 4T1 tumour-bearing mice treated with a single dose of free C1 (2.7 mg/kg) or C9 (2.1 mg/kg; 2.1 mg/kg C9 is equivalent of 2.7 mg/kg C1); PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGAC9 (2.1 mg/kg C9 equivalent/kg) nanoparticles; and cisplatin (1.2 mg/kg; 1.2 mg/kg cisplatin is equivalent to 2.7 mg/kg C1 and 2.1 mg/kg C9). Histology analysis revealed that in saline-treated 4T1 tumour-bearing mice, large metastatic foci were observed in the lungs, while small metastatic foci were observed in the liver (Fig. 6(a)). The large size and high number of metastatic foci present in the lungs (14 ± 3 foci) could possibly be related to the site-specific mechanism of metastasis, where the lung is reported to be the first site of breast cancer metastasis (Gao et al., 2008). Cisplatin at a single dose of 1.2 mg/kg significantly reduced lung metastasis by 35.7% (p < 0.0001, two-way ANOVA, Tukey’s post hoc test) compared to the saline-treated 4T1 tumour-bearing mice and inhibited metastasis to the liver (Fig. 6(b)). This observation indicates that although cisplatin reduces the tumour volume by approximately 41% compared to the saline control group, it did not completely inhibit the metastasis of the breast tumour. Significantly lower numbers of metastatic foci were observed in the lungs of C1- and C9-treated 4T1 mice (66.7% and 73% lower, respectively) than in the cisplatintreated groups (p < 0.0001, two-way ANOVA, Tukey’s post hoc test). Previous studies reported that other metal based complexes such as ruthenium arene complex inhibited only 36% of lung metastases compared to the control group, while its isostructural osmium(II) analogue did not inhibit lung metastasis (111% compared to control) in a murine

Page 18 of 35 transplantable mammary tumour that spontaneously metastasizes to the lungs (Bergamo et al., 2010). In PLGA-C1- and PLGA-C9-treated mice, no metastatic foci were observed in the lung and liver, suggesting that formulating tribenzyltin carboxylates into PLGA nanoparticles may improve the antimetastatic properties of these compounds and thus minimize the incidence of metastasis to these organs. These results are consistent with our earlier observations for in vitro cell migration, invasion and wound healing assays (Anasamy et al., 2017). 4.

CONCLUSIONS

The tribenzyltin carboxylates C1 and C9 demonstrated better tumour growth delay than cisplatin and doxorubicin in an in vivo setting. The enhanced tumour growth delay in mice treated with PLGA-C1 and PLGA-C9 nanoparticles compared with those treated with free drug and the control groups was consistent with the previous findings, in which their improved activity was linked to several factors, such as the increased rate of release in the tumoural acidic microenvironment, the drug’s sustained release action and the exploitation of the enhanced permeability and retention effect to achieve higher drug concentrations in tumour tissues (Bergamo et al., 2010; Gao et al., 2008; Mhlanga and Ray, 2015). In addition, the repeated doses (double and triple) of PLGA-C1 and PLGA-C9 nanoparticles further improved tumour growth suppression because in repeated drug administration, accumulation occurs, leading to prolonged circulation of the drug (Arnold et al., 2005). Moreover, C1, C9 and their respective PLGA nanoparticles demonstrated better antimetastatic properties than cisplatin. These data correlated well with our previous findings, where these complexes were able to reduce cancer cell motility, migration and invasion (Anasamy et al., 2017).

Page 19 of 35 From the process point of view, PLGA-based formulations are affordable and are relatively flexible to further customization either via surface modification techniques to produce stealth nanoparticles or by antibody/small targeting ligand conjugation to produce targetable nanoparticles (Kue et al., 2014; Kue et al., 2016; Voon et al., 2016). The likelihood of the PLGA nanoparticles to prolong the blood circulation of tribenzyltin complexes and the enhancement of drug accumulation at the tumour site can be ascertained by a biodistribution study. To develop these two tribenzyltin complexes or their derivatives as potential cancer chemotherapeutic drugs, it is pertinent to identify their direct therapeutic target(s) and elucidate their mechanism of action at the upstream cellular level, which triggers the previously reported downstream apoptotic pathway (Anasamy et al., 2017) and the current findings on the antitumour activities of these complexes. This understanding will allow further optimization of the active leads. DECLARATION OF INTEREST The authors declare that they have no conflict of interest. ACKNOWLEDGMENTS The work was supported by the Ministry of Higher Education, Malaysia (FP016-2018A). REFERENCES Acharya, S., Sahoo, S.K., 2011. PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv. Drug Deliv. Rev. 63, 170-183. https://doi.org/10.1016/j.addr.2010.10.008. Alama, A., Viale, M., Cilli, M., Bruzzo, C., Novelli, F., Tasso, B., Sparatore, F., 2009. In vitro cytotoxic activity of tri-n-butyltin(IV) lupinylsulfide hydrogen fumarate (IST-FS

Page 20 of 35 35) and preliminary antitumour activity in vivo. Invest. New Drugs. 27, 124-130. https://doi.org/10.1007/s10637-008-9148-x. Anasamy, T., Thy, C.K., Lo, K.M., Chee, C.F., Yeap, S.K., Kamalidehghan, B., Chung, L.Y., 2017. Tribenzyltin carboxylates as anticancer drug candidates: Effect on the cytotoxicity, motility and invasiveness of breast cancer cell lines. Eur. J. Med. Chem. 125, 770-783. https://doi.org/10.1016/j.ejmech.2016.09.061. Arnold, R.D., Mager, D.E., Slack, J.E., Straubinger, R.M., 2005. Effect of repetitive administration of doxorubicin-containing liposomes on plasma pharmacokinetics and drug biodistribution in a rat brain tumour model. Clin. Cancer Res. 11, 8856-8865. https://doi.org/10.1158/1078-0432.CCR-05-1365. Baalousha, M., Lead, J., 2012. Rationalizing nanomaterial sizes measured by atomic force microscopy, flow field-flow fractionation, and dynamic light scattering: Sample preparation, polydispersity, and particle structure. Environ. Sci. Technol. 46, 61346142. https://doi.org/10.1021/es301167x. Bergamo, A., Masi, A., Peacock, A., Habtemariam, A., Sadler, P.J., Sava, G., 2010. In vivo tumour and metastasis reduction and in vitro effects on invasion assays of the ruthenium RM175 and osmium AFAP51 organometallics in the mammary cancer model. J. Inorg. Biochem. 104, 79-86. https://doi.org/10.1016/j.jinorgbio.2009. Binder, S., 2013. Evolution of taxanes in the treatment of metastatic breast cancer. Clin. J. Oncol. Nurs. 17 Suppl, 9-14. https://doi.org/10.1188/13.cjon.s1.9-14. Bowerman, C.J., Byrne, J.D., Chu, K.S., Schorzman, A.N., Keeler, A.W., Sherwood, C.A., Perry, J.L., Luft, J.C., Darr, D.B., Deal, A.M., Napier, M.E., Zamboni, W.C., Sharpless, N.E., Perou, C.M., DeSimone, J.M., 2017. Docetaxel-loaded PLGA

Page 21 of 35 nanoparticles improve efficacy in taxane-resistant triple-negative breast cancer. Nano Lett. 17, 242-248. https://doi.org/10.1021/acs.nanolett.6b03971. Bulatovic, M.Z., Maksimovic-Ivanic, D., Bensing, C., Gomez-Ruiz, S., Steinborn, D., Schmidt, H., Mojic, M., Korac, A., Golic, I., Perez-Quintanilla, D., Momcilovic, M., Mijatovic, S., Kaluderovic, G.N., 2014. Organotin(IV)-loaded mesoporous silica as a biocompatible strategy in cancer treatment. Angew. Chem. Int. Ed. Engl. 53, 59825987. https://doi.org/10.1002/anie.201400763. Chen, M., Bertino, J.S., Berg, M.J., Nafziger, A.N., 2007. CHAPTER 21 - Pharmacological Differences between Men and Women, in: Atkinson, A.J., Abernethy, D.R., Daniels, C.E., Dedrick, R.L., Markey, S.P. (Eds.), Principles of Clinical Pharmacology, second ed. Academic Press, Burlington, pp. 325-338. Danhier, F., Ansorena, E., Silva, J.M., Coco, R., Le Breton, A., Préat, V., 2012. PLGAbased nanoparticles: An overview of biomedical applications. J. Controlled Release. 161, 505-522. https://doi.org/10.1016/j.jconrel.2012.01.043. Deryabin, D.G., Efremova, L.V., Vasilchenko, A.S., Saidakova, E.V., Sizova, E.A., Troshin, P.A., Zhilenkov, A.V., Khakina, E.E., 2015. A zeta potential value determines the aggregate’s size of penta-substituted[60]fullerene derivatives in aqueous suspension whereas positive charge is required for toxicity against bacterial cells. J. Nanobiotechnology. 13, 50. https://doi.org/10.1186/s12951-015-0112-6. Duan, F., Simeone, S., Wu, R., Grady, J., Mandoiu, I., Srivastava, P.K., 2012. Area under the curve as a tool to measure kinetics of tumour growth in experimental animals. J. Immunol. Methods. 382, 224-228. https://doi.org/10.1016/j.jim.2012.06.005.

Page 22 of 35 Etaiw, S.E.-d.H., Sultan, A.S., El-din, A.S.B., 2011. A novel hydrogen bonded bimetallic supramolecular coordination polymer {[SnMe3(bpe)][Ag(CN)2]·2H2O} as anticancer drug.

Eur.

J.

Med.

Chem.

46,

5370-5378.

https://doi.org/10.1016/j.ejmech.2011.08.041. Gaillard, P.J., Visser, C.C., de Boer, M., Appeldoorn, C.C., Rip, J., 2014. Blood-to-brain Drug Delivery Using Nanocarriers, Drug Delivery to the Brain. Springer Press., New York, pp. 433-454. Gao, D., Du, J., Cong, L., Liu, Q., 2008. Risk factors for initial lung metastasis from breast invasive ductal carcinoma in stages I–III of operable patients. Jpn. J. Clin. Oncol. 39, 97-104. https://doi.org/10.1093/jjco/hyn133. Göschl, S., Schreiber-Brynzak, E., Pichler, V., Cseh, K., Heffeter, P., Jungwirth, U., Jakupec, M.A., Berger, W., Keppler, B.K., 2017. Comparative studies of oxaliplatinbased platinum(IV) complexes in different in vitro and in vivo tumour models. Metallomics. 9, 309-322. https://doi.org/10.1039/c6mt00226a. Harper, B.W., Petruzzella, E., Sirota, R., Faccioli, F.F., Aldrich-Wright, J.R., Gandin, V., Gibson, D., 2017. Synthesis, characterization and in vitro and in vivo anticancer activity

of

Pt(IV)

derivatives

of

[Pt(1S,2S-DACH)(5,

6-dimethyl-1,10-

phenanthroline)]. Dalton Trans. 46, 7005-7019. https://doi.org/10.1039/c7dt01054. Kai, M.P., Keeler, A.W., Perry, J.L., Reuter, K.G., Luft, J.C., O'Neal, S.K., Zamboni, W.C., DeSimone, J.M., 2015. Evaluation of drug loading, pharmacokinetic behavior, and toxicity of a cisplatin-containing hydrogel nanoparticle. J. Controlled Release. 204, 7077. https://doi.org/10.1016/j.jconrel.2015.03.001.

Page 23 of 35 Koudelka, Š., Turánek‐ Knötigová, P., MaŠek, J., Korvasová, Z., Škrabalová, M., Plocková, J., Bartheldyová, E., Turánek, J., 2010. Liposomes with high encapsulation capacity for paclitaxel: Preparation, characterisation and in vivo anticancer effect. J. Pharm. Sci. 99, 2309-2319. https://doi.org/10.1002/jps.21992. Kue, C.S., Kamkaew, A., Lee, H.B., Chung, L.Y., Kiew, L.V., Burgess, K., 2014. Targeted PDT agent eradicates TrkC expressing tumours via photodynamic therapy (PDT). Mol. Pharm. 12, 212-222. https://doi.org/10.1021/mp500556. Kue, C.S., Kamkaew, A., Voon, S.H., Kiew, L.V., Chung, L.Y., Burgess, K., Lee, H.B., 2016. Tropomyosin receptor kinase C targeted delivery of a peptidomimetic ligandphotosensitizer

conjugate

induces

antitumour

immune

responses

following

photodynamic therapy. Sci. Rep. 6, 37209. https://doi.org/10.1038/srep37209. Kumari, A., Yadav, S.K., Yadav, S.C., 2010. Biodegradable polymeric nanoparticles-based drug

delivery

systems.

Colloids

Surf.,

B.

75,

1-18.

https://doi.org/10.1016/j.colsurfb.2009.09.001. Li, M., Tang, Z., Zhang, Y., Lv, S., Li, Q., Chen, X., 2015. Targeted delivery of Cisplatin by LHRH-peptide conjugated dextran nanoparticles suppresses breast cancer growth and metastasis. Acta. Biomater. 18, 132-143. https://doi.org/10.1016/j.actbio.2015.02.022. Lu, Y., Park, K., 2013. Polymeric micelles and alternative nano sized delivery vehicles for poorly

soluble

drugs.

Int.

J.

Pharm.

453,

198-214.

https://doi.org/10.1016/j.ijpharm.2012.08.042. Maeda, H., Bharate, G.Y., Daruwalla, J., 2009. Polymeric drugs for efficient tumourtargeted drug delivery based on EPR-effect. Eur. J. Pharm. Biopharm. 71, 409-419. https://doi.org/10.1016/j.ejpb.2008.11.010.

Page 24 of 35 Mattheolabakis, G., Taoufik, E., Haralambous, S., Roberts, M.L., Avgoustakis, K., 2009. In vivo investigation of tolerance and antitumour activity of cisplatin-loaded PLGAmPEG

nanoparticles.

Eur.

J.

Pharm.

Biopharm.

71,

190-195.

https://doi.org/10.1016/j.ejpb.2008.09.01. Mhlanga, N., Ray, S.S., 2015. Kinetic models for the release of the anticancer drug doxorubicin from biodegradable polylactide/metal oxide-based hybrids. Int. J. Biol. Macromol. 72, 1301-1307. https://doi.org/10.1016/j.ijbiomac.2014.10.038. Murdock, R.C., Braydich-Stolle, L., Schrand, A.M., Schlager, J.J., Hussain, S.M., 2008. Characterization of nanomaterial dispersion in solution prior to in vitro exposure using dynamic

light

scattering

technique.

Toxicol.

Sci.

101,

239-253.

https://doi.org/10.1093/toxsci/kfm240. Navakoski de Oliveira, K., Andermark, V., Onambele, L.A., Dahl, G., Prokop, A., Ott, I., 2014. Organotin complexes containing carboxylate ligands with maleimide and naphthalimide derived partial structures: TrxR inhibition, cytotoxicity and activity in resistant

cancer

cells.

Eur.

J.

Med.

Chem.

87,

794-800.

https://doi.org/10.1016/j.ejmech.2014.09.075. Öztürk-Atar, K., Eroğlu, H., Çalış, S., 2017. Novel advances in targeted drug delivery. J. Drug Targeting. 26, 633-642. https://doi.org/10.1080/1061186X.2017.1401076. Park, J.H., Saravanakumar, G., Kim, K., Kwon, I.C., 2010. Targeted delivery of low molecular drugs using chitosan and its derivatives. Adv. Drug Deliv. Rev. 62, 28-41. https://doi.org/10.1016/j.addr.2009.10.003. Saneja, A., Kumar, R., Singh, A., Dhar Dubey, R., Mintoo, M.J., Singh, G., Mondhe, D.M., Panda, A.K., Gupta, P.N., 2017. Development and evaluation of long-circulating

Page 25 of 35 nanoparticles loaded with betulinic acid for improved anti-tumour efficacy. Int. J. Pharm. 531, 153-166. https://doi.org/10.1016/j.ijpharm.2017.08.076. Sirajuddin, M., Ali, S., 2016. Organotin(IV) carboxylates as promising potential drug candidates in the field of cancer chemotherapy. Curr. Pharm. Des. 22, 6665-6681. https://doi.org/10.2174/1381612822666160906143249. ten Tije, A.J., Verweij, J., Loos, W.J., Sparreboom, A., 2003. Pharmacological effects of formulation vehicles : Implications for cancer chemotherapy. Clin. Pharmacokinet. 42, 665-685. https://doi.org/10.2165/00003088-200342070-00005. Varela-Ramirez, A., Costanzo, M., Carrasco, Y.P., Pannell, K.H., Aguilera, R.J., 2011. Cytotoxic effects of two organotin compounds and their mode of inflicting cell death on

four

mammalian

cancer

cells.

Cell.

Biol.

Toxicol.

27,

159-168.

https://doi.org/10.1007/s10565-010-9178-y. Verginadis, I.I., Karkabounas, S., Simos, Y., Kontargiris, E., Hadjikakou, S.K., Batistatou, A., Evangelou, A., Charalabopoulos, K., 2011. Anticancer and cytotoxic effects of a triorganotin compound with 2-mercapto-nicotinic acid in malignant cell lines and tumour

bearing

Wistar

rats.

Eur.

J.

Pharm.

Sci.

42,

253-261.

https://doi.org/10.1016/j.ejps.2010.11.015. Voon, S.H., Tiew, S.X., Kue, C.S., Lee, H.B., Kiew, L.V., Misran, M., Kamkaew, A., Burgess, K., Chung, L.Y., 2016. Chitosan-coated poly(lactic-co-glycolic acid)diiodinated boron-dipyrromethene nanoparticles improve tumour selectivity and stealth properties in photodynamic cancer therapy. J. Biomed. Nanotechnol. 12, 14311452. https://doi.org/10.1166/jbn.2016.2263.

Page 26 of 35 Wehbe, M., Malhotra, A.K., Anantha, M., Lo, C., Dragowska, W.H., Dos Santos, N., Bally, M.B., 2018. Development of a copper-clioquinol formulation suitable for intravenous use. Drug Delivery Transl. Res. 2018, 1-13. https://doi.org/10.1007/s13346-017-04557. Weiss, R.B., Donehower, R.C., Wiernik, P.H., Ohnuma, T., Gralla, R.J., Trump, D.L., Baker, J.R., Jr., Van Echo, D.A., Von Hoff, D.D., Leyland-Jones, B., 1990. Hypersensitivity

reactions

from

taxol.

J.

Clin.

Oncol.

8,

1263-1268.

https://doi.org/10.1200/jco.1990.8.7.1263. Xiong, Y., Jiang, W., Shen, Y., Li, H., Sun, C., Ouahab, A., Tu, J., 2012. A poly(γ, lglutamic acid)-citric acid based nanoconjugate for cisplatin delivery. Biomaterials. 33, 7182-7193. https://doi.org/10.1016/j.biomaterials.2012.06.071. Yallapu, M.M., Gupta, B.K., Jaggi, M., Chauhan, S.C., 2010. Fabrication of curcumin encapsulated PLGA nanoparticles for improved therapeutic effects in metastatic cancer cells. J. Colloid Interface Sci. 351, 19-29. https://doi.org/10.1016/j.jcis.2010.05.022. Yokoyama, M., Okano, T., Sakurai, Y., Ekimoto, H., Shibazaki, C., Kataoka, K., 1991. Toxicity and antitumour activity against solid tumours of micelle-forming polymeric anticancer drug and its extremely long circulation in blood. Cancer Res. 51, 32293236. TABLE LEGEND Table 1. Physicochemical characterisation of PLGA-NP, PLGA-C1 and PLGA-C9 nanoparticles.

FIGURE LEGENDS

Page 27 of 35 Fig. 1. Chemical structures of tri(4-fluorobenzyl)tin[(N,N-diisopropylcarbamothioyl sulfanyl]acetate (C1) and tribenzyltin isonicotinate (C9). Fig. 2. In vivo toxicity profile of (a) free C1, (b) free C9, and (c) cisplatin in BALB/c mice. C1, C9 and cisplatin were well tolerated at doses of 12.5 mg/kg, 8 mg/kg and 6 mg/kg respectively. The data represent the fraction change in body weight with two mice per treatment group. Fig. 3. In vivo antitumour efficacy studies of free C1 and free C9 in 4T1 tumour-bearing BALB/c mice. Mice treated with a single dose of free C1 (2.7 mg/kg) and C9 (2.1 mg/kg) showed delayed tumour growth compared to the cisplatin, doxorubicin and saline control groups. Mice treated with repeated doses of C1 (2.7 mg/kg, days 1 and 7)†† and C9 (2.1 mg/kg, days 1 and 7)†† showed better tumour growth delay compared to the single-dose administration. The vehicle control contained saline with 5% ethanol and 5% Cremophor EL. Data represent the mean tumour volume ± SEM (n=7). **** p < 0.0001 for saline and vehicle control vs C1 (2.7 mg/kg) or C9 (2.1 mg/kg) as well as saline and vehicle control vs C1 (2.7 mg/kg, days 1 and 7)†† or C9 (2.1 mg/kg, days 1 and 7)†† using two-way ANOVA. Fig. 4. In vivo toxicity profile of (a) PLGA-C1 and (b) PLGA-C9 in BALB/c mice. Encapsulation of C1 and C9 into PLGA nanoparticles increased their tolerated dose to 17.5 mg C1 equivalent/kg for PLGA-C1 and 12 mg C9 equivalent/kg for PLGA-C9. The data represent the fraction change in body weight with two mice per treatment group. (PLGA = poly(lactic-co-glycolic acid), NP = nanoparticle). Fig. 5. In vivo antitumour efficacy studies of PLGA-C1 and PLGA-C9 in 4T1 tumourbearing BALB/c mice. (a) PLGA-C1 administered at single doses of 2.7 mg C1 equivalent/kg and 5.4 mg C1 equivalent/kg, and repeated doses (5.4 mg C1 equivalent/kg,

Page 28 of 35 days 1 and 7)†† and (5.4 mg C1 equivalent/kg, days 1, 5 and 9)††† showed better tumour growth suppression than the free drug-treated mice. (b) PLGA-C9 administered at single doses (2.1 mg C9 equivalent/kg) and (4.2 mg C9 equivalent/kg) and repeated doses (4.2 mg equivalent C9/kg, days 1 and 7)†† and (4.2 mg equivalent C9/kg, days 1, 5 and 9)††† showed better tumour growth suppression than the free drug-treated mice. Triple-dose administration showed improved antitumour efficacy compared to the double-dose administration. The data represent the mean tumour volume ± SEM (n=7) for each treatment group. Tumour growth curves of free C1 (2.7 mg/kg) or C9 (2.1 mg/kg), saline and blank PLGA-NP treated groups are added for comparison. ***p < 0.001 for C1 (2.7 mg/kg) or C9 (2.1 mg/kg) vs PLGA-C1 (5.4 mg C1 equivalent/kg)††† or PLGA-C9 (4.2 mg C9 equivalent/kg)†††, respectively and ** p < 0.01 for C1 (2.7 mg/kg) or C9 (2.1 mg/kg) vs PLGA-C1 (5.4 mg C1 equivalent/kg)†† or PLGA-C9 (4.2 mg C9 equivalent/kg)††, respectively using two-way ANOVA. (PLGA = poly(lactic-co-glycolic acid)). Fig. 6. Histopathological analysis and quantification of metastatic foci. (a) Kidney, liver and lung histology profiles of healthy mice, untreated 4T1-tumour bearing mice and 4T1-tumour bearing mice treated with a single dose of free C1 (2.7 mg/kg) and C9 (2.1 mg/kg; 2.1 mg/kg C9 is equivalent to 2.7 mg/kg C1), PLGA-C1 (2.7 mg C1 equivalent/kg) and PLGAC9 (2.1 mg C9 equivalent/kg) nanoparticles and cisplatin (1.2 mg/kg, 1.2 mg/kg cisplatin is equivalent to 2.7 mg/kg C1 and 2.1 mg/kg C9). No evidence of metastasis was observed in the major organs extracted from PLGA-C1 and PLGA-C9 nanoparticle-treated mice. Yellow arrows indicate metastatic foci. (b) Quantification of metastatic foci in the kidney, liver and lung. Data represent the mean number of metastatic foci ± SEM (n=3). ****p < 0.0001 for 4T1 tumour-bearing mice vs C1 or C9 and C1 or C9 vs cisplatin treated group, **p < 0.01

Page 29 of 35 for C1 vs PLGA-C1 treated group and *p < 0.05 for C9 vs PLGA-C9 treated group, using two-way ANOVA. (PLGA = poly(lactic-co-glycolic acid)).

CREDIT AUTHOR STATEMENT Theebaa Anasamy: Conceptualization, Methodology, Investigation, Writing- Original draft preparation. Chee Chin Fei: Conceptualization, Methodology, Resources, WritingReviewing and Editing. Kiew Lik Voon: Conceptualization, Resources, Writing- Reviewing and Editing. Chung Lip Yong: Conceptualization, Writing- Reviewing and Editing, Supervision, Project Administration, Funding Acquisition.

Table 1. Physicochemical characterisation of PLGA-NP, PLGA-C1 and PLGA-C9 nanoparticles. Formulation % Particle PDI Zeta Loading Entrapment Yield PVA size (nm) Potential Efficiency Efficiency (%) (w/v) (mV) (%) (%) 136.7 ± 0.07 ± -14.2 ± 74.3 ± 1.5 NA NA PLGA-NP 0.3 0.03 0.3 3.3 183.7 ± 0.05 ± -14.7 ± 70.3 ± 1.5 13.4 ± 1.4 61.1 ± 3.7 PLGA-C1 0.8 0.01 0.1 5.1 163.2 ± 0.08 ± -14.8 ± 73.1 ± 1.5 12.2 ± 1.8 55.8 ± 4.3 PLGA-C9 1.2 0.01 0.2 6.8 Data are expressed as mean ± SD (n = 6). PLGA = poly(lactic-co-glycolic acid), NP = nanoparticles, PDI = polydispersity index, NA = not available.

fig. 1

Page 30 of 35

Page 31 of 35 fig. 2

Page 32 of 35 fig. 3

fig. 4

Page 33 of 35 fig. 5

fig.6

Page 34 of 35

fig. 6

Page 35 of 35 Graphical Abstract