Docetaxel-loaded polyglutamic acid-PEG nanocapsules for the treatment of metastatic cancer

    Docetaxel-loaded polyglutamic acid-PEG nanocapsules for the treatment of metastatic cancer Erea Borrajo, Raquel Abellan-Pose, Atenea Soto, Marcos Garcia-Fuentes, Noemi Csaba, Maria J. Alonso, Anxo Vidal PII: DOI: Reference:

S0168-3659(16)30494-1 doi: 10.1016/j.jconrel.2016.07.048 COREL 8405

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

4 April 2016 27 July 2016 28 July 2016

Please cite this article as: Erea Borrajo, Raquel Abellan-Pose, Atenea Soto, Marcos Garcia-Fuentes, Noemi Csaba, Maria J. Alonso, Anxo Vidal, Docetaxel-loaded polyglutamic acid-PEG nanocapsules for the treatment of metastatic cancer, Journal of Controlled Release (2016), doi: 10.1016/j.jconrel.2016.07.048

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Docetaxel-loaded Polyglutamic acid-PEG Nanocapsules for the Treatment of Metastatic Cancer

T

Erea Borrajo1, Raquel Abellan-Pose1, Atenea Soto, Marcos Garcia-Fuentes, Noemi

These authors contributed equally to this work.

SC R

1

IP

Csaba, Maria J. Alonso*, Anxo Vidal*

Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University

Corresponding authors: Maria J. Alonso Fernández and Anxo Vidal

MA

*

NU

of Santiago de Compostela, 15706 Campus Vida, Santiago de Compostela, Spain.

D

E-mail adresses: [email protected] and [email protected]

TE

KEY WORDS: Nanomedicine, nanocapsules, nanoparticles, drug delivery, anti-cancer,

AC

CE P

oncologicals.

1

ACCEPTED MANUSCRIPT ABSTRACT The design of nanomedicines with suitable physicochemical characteristics for the

T

lymphatic targeting of drugs is critical in order to reach the lymph nodes, where

IP

metastatic cells often accumulate. Based on the known effect of particle size and surface

SC R

hydrophilicity on the capacity of nanocarriers to reach the lymph nodes, here we report the formation and characterization of 100 nm polyglutamic acid-polyethylene glycol (PGA-PEG) nanocapsules together with the assessment of their potential for the

NU

treatment of cancer with lymphatic metastatic spread. To this purpose, we first studied

MA

the biodistribution of fluorescently labeled PGA-PEG nanocapsules (100 nm), following, either intravenous or subcutaneous administration. The results confirmed the accumulation of nanocapsules in the lymphatic system, especially upon subcutaneous

TE

D

administration. Next, we evaluated the efficacy and toxicity of the docetaxel-loaded nanocapsules in an orthotopic lung cancer model that metastasizes to the lymph nodes.

CE P

As expected from the rational design, DCX-loaded PGA-PEG nanocapsules exhibited a greatly enhanced antitumoral efficacy and a reduced toxicity when compared with the

AC

commercial formulation Taxotere®. Furthermore, the administration of DCX-loaded PGA-PEG nanocapsules resulted in the practical elimination of the metastatic load in the mediastinal lymph nodes, whereas the treatment with the commercial formulation had a minor effect. Overall, these findings underscore the potential of PGA-PEG nanocapsules for the delivery of anticancer drugs to both, the tumor tissue and the metastatic lymph nodes. Therefore, they represent a promising therapy for the treatment of lung metastatic cancer.

2

ACCEPTED MANUSCRIPT 1

INTRODUCTION

According to the predictions from the World Health Organization, the number of new

T

cases is expected to rise by about 70% over the next two decades worldwide [1]. Lung

IP

cancer is the most common cancer and the leading cause of cancer deaths. Despite the

SC R

advances in early detection techniques and significant improvement in the surgical procedures, the survival rate for lung cancer is still very low [2]. From the clinical point of view, one of the major problems of lung cancer is that, at the moment of diagnosis,

NU

the tumor has already disseminated to the lymph nodes, aggravating patient prognosis

MA

[3]. Because of the well-known role of the lymphatic system in tumor cell spreading [4], the selective delivery of anticancer drugs, not only to the tumor but also to the draining lymph nodes, has been seen as a therapeutic strategy to control metastatic spreading and

TE

D

reduce systemic toxicity [5]. In this regard, a number of reports have already shown the capacity of different nanocarriers, including micelles [6–9], nanoparticles [10–13] and

CE P

nanocapsules [14] to improve the delivery of anticancer drugs to the draining lymph nodes. The results of these studies have underlined the enhanced therapeutic efficacy of

AC

the anticancer drug associated to the nanocarriers, when compared to the corresponding free drug solutions. In addition, the knowledge generated in this field has led to the conclusion that particle size and the surface hydrophilicity may influence the lymphatic distribution of the nanocarriers [5,15]. Namely, studies have shown that nanocarriers with a particle size close to 100 nm [16], negative zeta potential [17] and a hydrophilic surface [16,18] can migrate through the interstice, reach the lymphatics and accumulate into the lymph nodes, following subcutaneous administration (SC). It has also been reported that, following intravenous administration (IV), nanocarriers of very small particle size (usually less than 100 nm) may extravasate from the bloodstream to the interstice and be drained by the lymphatic system [7,8,19,20]. 3

ACCEPTED MANUSCRIPT In this context, our group has developed PGA-PEG nanocapsules, with the capacity to encapsulate hydrophobic drugs [21-23] and with a potential affinity for the lymphatic

T

system. Following IV administration to mice, these nanocapsules led to an increased

IP

efficacy of the associated drugs, together with a reduction of their systemic toxicity,

SC R

when compared to the corresponding reference drug formulations. This enhanced efficacy/toxicity balance was attributed to the capacity of the nanocapsules to modify the biodistribution of the encapsulated drugs. More recently, we developed ~100 nm

NU

PGA-PEG nanocapsules, which due to their small size, were supposed to accumulate in

MA

the lymphatics [23]. Indeed, following SC administration, these small 100 nmnanocapsules were drained to the lymph nodes faster than the larger ones (200 nm). Taking this information into consideration, the aim of the present work was to further

TE

D

investigate the lymphotargeting properties of ~100 nm PGA-PEG nanocapsules, and to study the antitumoral efficacy and toxicity of docetaxel-loaded nanocapsules in a

2.1

MATERIALS AND METHODS

AC

2

CE P

metastasizing orthotopic lung cancer model.

Chemicals

Miglyol® 812 was donated by Sasol Germany GmbH (Germany). The surfactant Epikuron® 145V was donated by Cargill (Spain). Hexadecyltrimethylammonium bromide (CTAB) and docetaxel (DCX) were obtained from Sigma-Aldrich (Spain). Polyglutamic acid-polyethylene glycol (PGA-PEG; Mw 35 kDa), a diblock copolymer of poly-L-glutamic acid (Mw=15 kDa) and PEG (Mw=20 kDa), was supplied by Alamanda Polymers, Inc. (USA). The near infrared dye 1,1′-dioctadecyl-3,3,3′,3′tetramethylindodicarbocyanine perchlorate (DiD) was purchased from Molecular

4

ACCEPTED MANUSCRIPT Probes-Invitrogen (USA). Taxotere® was kindly provided by the Medical Oncology Department of the University Clinical Hospital of Santiago de Compostela. Preparation and characterization of blank nanocapsules

T

2.2

IP

Polyglutamic acid-polyethylene glycol (PGA-PEG) nanocapsules were prepared by a

SC R

solvent-displacement technique as previously published [23]. Briefly, 7.5 mg of Epikuron® 145V were dissolved in 0.4 ml of ethanol. Next, 31.25 µl of Miglyol ® 812 and 0.1 ml of a CTAB ethanol solution (15 mg/ml) were added and mixed by vortex.

NU

After adding 9.5 ml of acetone, this organic phase was dropped as 250 µl aliquots every

MA

15 seconds into 20 ml of an aqueous solution of PGA-PEG (0.25 mg/ml), under magnetic stirring. This induced the spontaneous formation of the nanocapsules. The organic solvents were removed and the formulation was concentrated to 10 ml by

TE

D

evaporation under vacuum (Buchi Labortechnik AG, Flawil, Switzerland). The nanocapsules were isolated by ultracentrifugation in Herolab® tubes (Herolab GmbH,

CE P

Germany) at 84035 RCF for 1 hour at 15°C (OptimaTM L-90K Ultracentrifuge, Beckmann Coulter; Fullerton, CA).

AC

The size and polydispersity index of nanocapsules were analyzed by photon correlation spectroscopy (PCS) after appropriate dilution with ultrapure water. Each analysis was performed in triplicate at 25°C with an angle detection of 90°. The zeta potential was determined by laser Doppler anemometry (LDA) with the same sample. The PCS and LDA analysis were performed using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). 2.3

Preparation and characterization of fluorescently-labeled nanocapsules

Nanocapsules were fluorescently labeled by adding an aliquot of a stock solution of 2.5 mg/ml DiD in ethanol to the organic phase during their preparation. The nanocapsules were recovered by following the nanocapsule isolation procedure previously described. 5

ACCEPTED MANUSCRIPT DiD encapsulation efficiency was determined indirectly by the difference between the total amount of fluorescent probe in the formulation and the free dye quantified in the

T

infranatant after nanocapsules isolation. DiD was quantified by UV spectrophotometry

IP

at λ=646 nm. For the in vivo administration, the amount of DiD encapsulated in the

SC R

nanocapsules was also directly quantified by the same method, measuring the absorbance of an aliquot of isolated nanocapsules diluted 1:200 with acetonitrile. 2.4

Animals

NU

Female severe combined immunodeficiency (SCID) mice weighing about 20-25 g and

MA

8-12 weeks old were used for in vivo studies (Harlan Laboratories, Inc). The animals were acclimatized for at least 1 week before experimentation; they were housed in under SPF conditions in ventilated polypropylene cages at an average temperature of 22

TE

D

˚C, with exposure to 12 hours of light and 12 hours of darkness each day. All mice received a standard laboratory diet of food and water ad libitum. All experiments were

CE P

conducted under approval of the Santiago de Compostela University Bioethics Committee and in compliance with the Principles of Laboratory Animal Care according

AC

to Spanish national law (RD 53/2013). Humane endpoints were established on the basis of animal welfare criteria based on clinical signs of pain, distress and discomfort previously approved by the Animal Research Ethics Committee. 2.5

Biodistribution studies

Healthy non-tumor-bearing female SCID mice were injected intravenously in the tail vein or subcutaneously in the interscapular region with a suspension of 100 µl of DiDlabeled nanocapsules (nanocapsules concentration= 13 mg/ml, DiD concentration= 87 µg/ml). Biodistribution of the nanocapsules was assessed with an In Vivo Imaging System (IVIS, Living Image® System, Caliper Life Sciences, Hopkinton, MA).

6

ACCEPTED MANUSCRIPT For non-invasive tests, mice were anesthetized with 4% isoflurane inhalation. Once placed in the acquisition chamber, the anesthesia was maintained with a 2% air-

T

isofluran mixture during the in vivo acquisition. To compare the nanocapsules

IP

distribution on a semi-quantitative basis, mice were sacrificed at different time points

SC R

(6, 24 and 48 hours) after the administration, and the excised tissues, which included liver, lungs, spleen, kidneys, as well as axillary, cervical, inguinal, mediastinal and mesenteric lymph nodes, were compared with organs from non-injected animal as

NU

negative controls. Semi-quantitative data of the nanocapsule biodistribution were

MA

obtained from the fluorescence images by drawing regions of interest and using identical illumination settings. Organ fluorescence was quantified through mean radiance values.

D

Preparation of docetaxel-loaded nanocapsules

TE

2.6

PGA-PEG nanocapsules loaded with DCX (DCX nanocapsules) were prepared by the

CE P

solvent displacement technique [21], conveniently adapted to obtain a particle size around 100 nm [23]. Namely, 75 µl of a DCX stock ethanol solution (20 mg/ml) were

AC

added to 7.5 mg of Epikuron® 145V in 0.325 ml of ethanol. After mixing, 31.25 µl of Miglyol® 812, 0.1 ml of an ethanol solution of CTAB (15 mg/ml) and 9.5 ml of acetone were also incorporated. This organic phase was added drop wise (250 µl every 15 seconds) to 20 ml of a PGA-PEG aqueous solution (0.25 mg/ml) under magnetic stirring, which led to the immediate formation of nanocapsules. The solvents of the resulting suspension were evaporated under vacuum (Buchi Labortechnik AG, Flawil, Switzerland) to a final volume of 4 ml. Finally, nanocapsules were purified and concentrated by ultracentrifugation in Herolab® tubes (Herolab GmbH, Germany) at 84035 RCF for 1 hour at 15°C (OptimaTM L-90K Ultracentrifuge, Beckmann Coulter;

7

ACCEPTED MANUSCRIPT Fullerton, CA) and centrifugation in Amicon® Ultra-4 ML 10K centrifugal filter devices (Merck Millipore, Germany) at 7500 RCF, 20°C.

T

The DCX encapsulation efficiency was determined directly by measuring the drug in

IP

the concentrated isolated nanocapsules. To quantify the DCX, an aliquot was diluted in

SC R

acetonitrile 1:1200, centrifuged for 20 minutes at 4000 RCF and analyzed by high performance liquid chromatography (HPLC). DCX was quantified by a slightly modified version of the method described by Lee et al. [24]. The HPLC system

NU

consisted of an Agilent 1100 Series Instrument equipped with a UV detector set at 227

MA

nm and a reverse phase Zorbax Eclipse XDB-C8 column (4.6 x 150 mm id., pore size 5 µm Agilent USA). The mobile phase was a mixture of 0.1% v/v ortophosphoric acid and acetonitrile (55:45 v/v) and the flow rate was 1 ml/minute. The standard calibration

2.7

TE

D

curves of DCX were linear in the range of 0.2-2 µg/ml (r2 = 0.999). In vivo toxicity studies

CE P

The toxicity of DCX nanocapsules and of the commercial formulation Taxotere® were compared after IV administration of two different doses, 30 and 60 mg/kg, for 5

AC

consecutive days (5 and 10 mg/day, respectively) in SCID non tumor-bearing mice. Animals were subjected to detailed clinical observation and weighted daily during the duration of the study (15 days) and those showing signs of extreme weakness were sacrificed. The toxicity was quantified for all groups by comparing the maximum tolerated dose (MTD) defined as the maximum DCX dose resulting in less than 20% body weight loss and no lethality. Complete necropsy and histopatholoogical analysis on animal tissues were performed at sacrifice. 2.8

Orthotopic lung cancer model

The antitumor efficacy of DCX nanocapsules was assessed in an orthotopic lung cancer model that metastasizes to the mediastinal lymph nodes, as adapted from Cui et al. [25]. 8

ACCEPTED MANUSCRIPT Briefly, to generate this model, a suspension of 1x106 luciferase expressing non-small cell lung carcinoma cells (A549Luc cell line) in PBS (50 µl) was injected through the

T

intercostal space into the left lung of SCID mice (8-10 weeks old, 20-25 g). During this

IP

procedure, mice were anesthetized by 4% isoflurane inhalation. To follow tumor

SC R

growth, at selected time-points tumor-bearing animals were injected intra-peritoneally with Luciferin at a dose of 150 mg/kg and then anesthetized by isofluorane inhalation. Under anesthesia, images were taken over the left side of the mice using an IVIS®

NU

Spectrum System that allowed for a quantitative monitoring of both the primary tumor

MA

growth and the cancer cell dissemination. Based on the expression level of A549luc cell line we optimized exposure time (1 second), f-stop (2) and pixel binning (medium). A photographic image of the animal under white light was taken and a quantitative

TE

D

bioluminescent signal was overlaid on this image. The bioluminescent signal was expressed in photons per second and displayed as an intensity map. Luminescence from

CE P

the cells could be measured at the primary tumor using a region of interest tool. To support quantitation, the system measures dark charge during down-time and runs a

AC

self-calibration during initialization. Photon eflux detected from cancer cells is directly proportional to the number of living cells present in the tumor. Thus, the tumor volume is measured by the photon eflux which comes from cancer cells and differences in bioluminescence signal are a direct indication of the differences in tumor size. The presence of tumor cells in the lungs was confirmed by histological analysis with haematoxylin-eosin staining. At the end of the in vivo experiment or at appropriate humane endpoints, mice were sacrificed and proteins were extracted from different organs to quantify luciferase activity. Organs were homogenized in DIP buffer (50 mM pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% Tween-20, 10 mM β-Glycerophosphate, 1 mM 9

ACCEPTED MANUSCRIPT sodium orthovanadate, 0.1 M PMSF, 0.1 M NaF and protease inhibitor cocktail) using a tissue homogenizer. After centrifugation at 15000 RCF for 15 minutes, supernatants

T

were quantified by a Bradford colorimetric method and luminescence was measured

IP

using a Lumat BL 9507 luminometer (Berthold Technologies). Results were expressed

2.9

SC R

as relative luminescence units (RLU) per µg of extracted protein. In vivo antitumor efficacy study

The efficacy studies were carried out using DCX nanocapsules and Taxotere® as

NU

controls. Mice were randomly assigned to experimental and control groups (n=6). The

MA

study was performed twice with two different experimental designs. First, we confirmed the capacity of the treatments to block tumor development. Four groups of tumorbearing SCID mice were defined: i) control mice without treatment, ii) mice treated

TE

D

with control blank PGA-PEG nanocapsules, iii) mice treated with Taxotere® (20 mg/kg of DCX) and iv) mice treated with DCX nanocapsules (20 mg/kg of DCX). The

CE P

different formulations were administered intravenously by injecting 10 mg/Kg of DCX at 5 and 20 days after tumor cell implantation. The concentration of the nanocapsules

AC

administered was ≈120 mg/ml per injection. Tumor evolution was followed noninvasively by an IVIS® Spectrum System. At the end time point (37 days) the animals were sacrificed because control mice showed evidence signs of morbidity, and protein from lungs and mediastinal nodes was extracted for luciferase activity quantification as described above. In a second experimental design, the efficacy of DCX nanocapsules and Taxotere® was compared in an advanced stage cancer with metastasis. Five groups of tumor-bearing SCID mice were established according to the total dose of DCX: i) control mice without treatment, ii) mice injected with Taxotere® (10 mg/kg), iii) mice injected with Taxotere® (20 mg/kg), iv) mice injected with DCX nanocapsules (10 mg/kg) and v) 10

ACCEPTED MANUSCRIPT mice injected with DCX nanocapsules (20 mg/kg). The treatments were administered intravenously by injection at days 20 and 27 after tumor cell implantation. For the group

T

iv) and v) the concentration of nanocapsules administered were ≈ 90 mg/ml and 120

IP

mg/ml per injection, respectively. Body weight was measured weekly, the animals were

SC R

monitored daily for clinical signs of stress for the duration of the studies, and tumor evolution was followed by an IVIS® Spectrum System. According to the health conditions of the animals receiving or not receiving treatment, two different end time

NU

points were defined, days 37 and 47 after tumor cell injection. At these time points,

from lungs and mediastinal nodes. 2.10 Survival study

MA

surviving animals were sacrificed for measuring luciferase activity in protein extracts

TE

D

For the survival study, the initial point for the survival monitorization was the starting date of the treatment (i.e. twenty days after the injection of the tumoral cells in the lung)

CE P

and the end point was determined by the death of the animal during twenty-seven days. Five groups of tumor-bearing SCID mice were established according to the total dose of

AC

DCX: i) control mice without treatment, ii) mice injected with Taxotere® (10 mg/kg), iii) mice injected with Taxotere® (20 mg/kg), iv) mice injected with DCX nanocapsules (10 mg/kg) and v) mice injected with DCX nanocapsules (20 mg/kg). The treatments were administered intravenously by injection at days 20 and 27 after tumor cell implantation. To estimate treatment efficiency survival data were analyzed by the Kaplan-Meier method, using the Log-rank test (Mantel-Cox test). 2.11 Statistical analysis A Mann-Whitney Test was applied to examine the differences in the distribution and efficacy studies performed after treatment with either DCX nanocapsules or Taxotere®. The mean ± SEM was determined for each treatment group. The differences were 11

ACCEPTED MANUSCRIPT considered significant for *p < 0.05, and very significant for *p < 0.01. All the

Preparation and characterization of nanocapsules

SC R

3.1

RESULTS

IP

3

T

statistical analysis were carried out with GraphPad Prism Version 5.0 software.

PGA-PEG nanocapsules, prepared by the solvent displacement technique were loaded with the fluorescent marker DiD in order to study their biodistribution by in vivo

NU

fluorescence imaging. The nanocapsules were also loaded with the anticancer drug

MA

DCX for the assessment of their efficacy in an orthotopic lung cancer model. The results presented in Table 1 indicate the encapsulation of either DiD or DCX did not

TE

D

significantly modified the physicochemical properties of the nanocapsules.

CE P

Table 1. Physicochemical characteristics of blank, DiD-loaded and DCX-loaded PGAPEG nanocapsules. PdI: polydispersity index. Data represents mean ± SEM; n=3.

Size (nm)

PdI

ζ Potential (mV)

-

116 ± 5

0.1

-29 ± 5

DiD

107 ± 9

0.1

-36 ± 4

DCX

122 ± 2

0.1

-34 ± 5

AC

Loading

3.2

Biodistribution by fluorescence imaging

As indicated, the ability of DiD-loaded PGA-PEG nanocapsules to reach the lymphatic system was assessed by whole body non-invasive fluorescence imaging in a semiquantitative fashion [26]. This dye, DiD, has the advantage of being weakly fluorescent in water but highly fluorescent at lipid/water interfaces. It is also photostable 12

ACCEPTED MANUSCRIPT and has low interference with the animal tissues autofluorescence [27]. In a previous biodistribution study [23], we observed a high and persistent accumulation of

T

fluorescence at the injection site after SC administration, which is consistent with

IP

previous observations by others for liposomes [28]. This accumulation of cytotoxic

SC R

drugs such as docetaxel in a sc. tissue reservoir would result in a high toxic local effect, so the objective of this study was to compare this biodistribution pattern with the one obtained upon IV administration, and specifically analyze the fluorescence

NU

accumulation in the lymph nodes of healthy mice. The results showed that following IV

MA

administration to mice, PGA-PEG nanocapsules were accumulated mainly in the MPSrelated organs (liver, heart, lung, kidney and spleen) at all-time points (Figure 1). At 6 and 24 hours post-administration, the accumulation in these organs was significantly

TE

D

higher than that observed upon SC administration. However, at 48 hours postadministration, significant differences were only found in liver and spleen. Similarly,

CE P

the access of the nanocapsules to the lymphatic system (mesenteric, inguinal and mediastinal nodes) was higher following IV administration than SC administration

AC

(Figure 1A, 1B), although these differences become smaller over the time (Figure 1C). Based on these results, and taking into account the potential local damage that DCX might cause if released in the subcutaneous tissue, the IV route was selected for the subsequent in vivo studies.

13

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 1. Comparative biodistribution of DiD-loaded PGA-PEG nanocapsules injected IV and SC. Radiance (Photons/sec/cm2/sr x 107) in excised organs at (A) 6, (B) 24 and (C) 48 hours after injection. Data represents mean ± SEM; n=4. Mann Whitney test ((*) p <0.05, (**) p < 0.01). Mann Whitney test ((*) p <0.05, (**) p < 0.01), (***) p < 0.001).

14

ACCEPTED MANUSCRIPT 3.3

Toxicological evaluation

The systemic toxicity associated with DCX nanocapsules and the commercial

T

formulation Taxotere® used as reference was evaluated in non-tumor bearing SCID

IP

mice by IV administration. The maximum tolerated dose (MTD) range reported in the

SC R

literature for DCX in its commercial formulation Taxotere® is 15 to 40 mg/kg in inmunodeficient mice with heterotopic xenograft [29]. In our experiment, both treatments were administered for 5 consecutive days to achieve total doses of 30 or 60

NU

mg/kg. The body weight for mice treated with 30 mg/kg of Taxotere® or with any dose

MA

of DCX nanocapsules barely changed for the duration of the study, which was prolonged up to 10 days after the last administration. However, when mice were treated with 60 mg/kg of encapsulated DCX their body weight loss was less than 5% and no

TE

D

toxicity signs were evidenced during the experiment. On the contrary, the mice treated with Taxotere® at the same dose (60 mg/kg) suffered a 27.2% loss of body weight

CE P

(Figure 2) and presented signs of toxicity, such as piloerection, tremor and weakness, although no mortality was observed. No clinical signs or behavioral alterations were

AC

recorded in the other experimental groups. A histopathological analysis was performed upon sacrifice and no histological abnormalities were found in liver, kidney, brain, heart or skin from NCs-treated animals (data not shown). These results indicate that DCX formulated in PGA-PEG nanocapsules exhibits a significantly lower toxicity that the commercial formulation.

15

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

In vivo antitumor efficacy

CE P

3.4

TE

D

MA

Figure 2. Effect of Taxotere and DCX-loaded PGA-PEG nanocapsules (DCX nanocapsules) on mice body weight over 15 days after the first administration. The formulations tested were: Taxotere and DCX loaded PGA-PEG nanocapsules. The tested doses were 30 and 60 mg/kg for Taxotere (TXT 30 and TXT 60, respectively) and DCX nanocapsules (NCs 30 and NCs 60, respectively). Data represents mean ± SEM.; n=3. Mann Whitney test ((**) p < 0.01).

This study was aimed at evaluating the capacity of DCX nanocapsules to inhibit tumor

AC

growth and, more importantly, to prevent cancer cells from spreading from the primary tumor. For this purpose, an A549luc orthotopic lung cancer model with characteristic tumor spreading to the mediastinal lymph nodes was used. In this model, metastasis in the lymph nodes could be unequivocally detected 13 days after the injection of tumor cells (Supplemental material, Figure 1). To test the antitumoral activity of the DCX nanocapsules, the effect of this formulation and appropriate controls was evaluated both, in the primary tumor and in the lymph nodes, following IV administration. In all experiments, untreated mice were used as negative controls and Taxotere® treated mice as positive control. After the orthotopical implantation of A549luc cells, tumor-bearing animals were treated 5 and 20 days later 16

ACCEPTED MANUSCRIPT with 20 mg/kg of DCX either as the commercial Taxotere® formulation or as DCX nanocapsules. The tumor growth was monitored in vivo by measuring the

T

bioluminescence of the tumor cells in the lung using the IVIS® Spectrum System. The

IP

results showed that, in the case of untreated animals tumor size increased exponentially

SC R

throughout the experiment. Similarly, in the case of mice treated with blank nanocapsules tumor growth was comparable to that of the control group. In contrast, the groups of mice receiving either Taxotere® or DCX-loaded nanocapsules showed no

NU

tumor growth during the experiment (Figure 3A-B).

MA

The comparable antitumoral effect of Taxotere® and DCX nanocapsules was confirmed when animals were sacrificed and luciferase activity determined. Lungs from the untreated or blank nanocapsule-treated groups showed a high tumor burden, whereas

tumor load (Figure 3C).

TE

D

mice treated with Taxotere® or DCX nanocapsules showed a significant reduction in

CE P

Interestingly, luciferase activity in mediastinal nodes from the four experimental groups was correlated to that of the primary tumor (Figure 3D). All these results suggest that

AC

DCX nanocapsules are as effective as Taxotere® in terms of controlling both, primary tumor growth and lymphatic metastasis, when the treatment is administered at an early stage of tumor development. The reduced number of cancer cells found in the lymph nodes in DCX-treated animals could be due either to the direct effect of DCX on the metastasis or be a secondary effect, a consequence of the decrease in cell spreading due to the reduction in the size of the primary tumor.

17

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

Figure 3. (A) Tumor growth in mice treated with different DCX formulations and controls, measured by in vivo luminescence. Arrows represent DCX administration time points in the study. (B) Images showing tumor cell loading in lung and mediastinal nodes as measured by ex-vivo bioluminescence images with an IVIS® Spectrum. (C) Quantification of luciferase activity (average RLU per µg of protein measured for luciferase activity) measured ex vivo (C) in lung and (D) in mediastinal lymph nodes. Experimental groups were: Taxotere® (TXT 20) and DCX nanocapsules (NCs 20) at 20 mg/kg DCX dose, blank nanocapsules (Blank NCs) and no treatment (Control). Data represents mean ± SEM; n=5. Mann Whitney test ((*) p <0.05, (**) p < 0.01) relative to control group.

To address this issue, the formulations were also tested at a more advanced tumor stage, when lymph node metastasis was already evident. Mice were treated with Taxotere® or DCX-loaded nanocapsules at days 20 and 27 after the injection of cancer cells. Two doses of DCX were evaluated in this experiment, 10 and 20 mg/kg. Two endpoints were also defined: animals were sacrificed at 37 or 47 days after tumor cell injection according to the different health conditions observed for the different groups. At day 37, untreated animals (control) had to be sacrificed because of their high tumor burden, 18

ACCEPTED MANUSCRIPT whereas mice treated with either Taxotere® or DCX-loaded nanocapsules showed a similar reduction in tumor growth (Figure 4A). At day 47, the groups treated with the

T

Taxotere® 10 mg/kg formulation started to show an exponential tumor growth, while the

IP

rest of the experimental groups presented an inhibitory effect on the tumor growth. The

SC R

Kaplan-Meier survival plot illustrates the capacity of all DCX formulations, with the

CE P

TE

D

MA

NU

exception of Taxotere® 10 mg/kg, to control tumor growth (Figure 4B).

Figure 4. (A) Tumor growth in mice treated with different DCX formulations and doses as measured using a bioluminescence imaging system in vivo IVIS® Spectrum. Arrows represent the administration time points for the experiment. Data represents mean ±

AC

SEM; n=6. (B) Kaplan-Meier survival plots. The formulations tested were: Taxotere® and DCX loaded PGA-PEG nanocapsules (DCX nanocapsules). The DCX doses tested were 10 and 20 mg/kg. Survival analysis by Log-rank (Mantel Cox) Test: Taxotere® 10 mg/kg (TXT 10) vs DCX nanocapsules (NCs 10) 10 mg/kg, p= 0.0096, Significative (**); Taxotere® 20 mg/kg (TXT 20) vs NC DCX 20 mg/kg (NCs 20), p= 0.3613, nonsignificative.

For a more detailed quantitative analysis on the tumor and the lymph nodes, luciferase activity was measured in organs from sacrificed mice to measure cancer cell load. At 37 days, all DCX formulations with 20 mg/kg were effectively reduced the tumor burden compared to control (Figure 5A). Similar results were found when the mediastinal 19

ACCEPTED MANUSCRIPT lymph nodes were analyzed (Figure 5B). At day 47, cancer cell loads in the lungs and in the mediastinal nodes were significantly lower for mice treated with DCX

T

nanocapsules as compared to those treated with Taxotere®, irrespective of the DCX

IP

dose (Figure 5 A, B).

SC R

When we compared luciferase activity values in the mediastinal nodes of the treated groups with the values obtained before treatment (day 19 after the injection of the tumor cells), we observed that all DCX treatments were able to maintain the number of

NU

metastatic cells for up to day 37. However, these numbers increased significantly during

MA

the following days and, at day 47, only the DCX-loaded nanocapsules at 20 mg/kg prevented the metastatic growth. Interestingly, in this group, the metastatic cell load was significantly lower than the one found before treatment (Figure 5C). These results show

TE

D

that DCX nanocapsules have a higher and a more prolonged effect on lymphatic

AC

CE P

metastasis than Taxotere®.

20

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 5. Luciferase activity (average RLU per µg of protein measured for luciferase activity) quantified ex-vivo (A) in the lungs and in (B) mediastinal lymph nodes, 37 and 47 days after the injection of the tumor cells. (C) Comparative graphical representation of the ex-vivo luciferase activity quantified in mediastinal lymph nodes before the treatment (T0, day 19) versus the end time at 37 and 47 days after the injection of the tumor cells. Dotted line represents the tumor cell loading before treatments (day 19). Mice were treated with Taxotere® 10 and 20 mg/kg total dose (TXT 10 and TXT 20, respectively) and PGA-PEG nanocapsules at 10 and 20 mg/kg DCX total dose (NCs 10

21

ACCEPTED MANUSCRIPT and NCs 20, respectively). Non treated animals were used as control. Data represents mean ± SEM; n=5. Mann Whitney test ((*) p <0.05, (**) p < 0.01)).

DISCUSSION

T

4

IP

To improve the prognosis associated with a cancer diagnosis and to prevent the

SC R

metastatic spread are two of the driving forces behind the design of new oncological therapies. The nanomedicine field can greatly contribute to these goals through the design of nanocarriers with optimal physicochemical characteristics for the lymphatic

NU

targeting of drugs. In previous reports, we have shown the potential polymer

MA

nanocapsules have as carriers for the delivery of anticancer drugs [21,22,30–36]. Among these nanocarriers, we have found that those with a PGA-PEG shell exhibited a

D

comparable antitumor activity and lower toxicity than the commercial formulation

TE

Taxotere® [36]. Subsequently, and considering previous work that underlined the importance of a small size for enhancing the delivery of nanocarriers to the lymph nodes

CE P

[16,28], we optimized the PGA-PEG nanocapsules preparation technique and obtained nanocapsules ~100 nm in size [23]. As expected, following SC administration, this

AC

reduction in size facilitated their migration through the interstitial channels and, thus, their lymphatic drainage. However, although this route of administration is traditionally the most widely used to study the lymphatic targeting of drugs or diagnostic agents, it may present certain limitations for the lymphatic targeting of anticancer drugs, such as local toxicity [5]. For this reason, in this work, we have considered another administration route and compared the lymphatic access of PGA-PEG nanocapsules upon IV and SC administration to healthy mice. The results indicated that, in line with recently reported data [20], following IV administration, fluorescent PGA-PEG nanocapsules accumulate in the MPS organs (liver, lung, spleen) although a significant fluorescent signal was also observed in the lymph nodes. Contrary to the expectation, 22

ACCEPTED MANUSCRIPT the fluorescence levels in all organs were lower following SC administration (Figure 1). This limited access of the nanocapsules to the lymphatic system could be attributed

T

to the fact that the injection in the interscapular region is less effective than in other

IP

places (such as the flank or the footpad), where the organization of the subcutaneous

SC R

tissue promotes an increase in the interstitial fluid pressure and thus the lymphatic drainage [20,37]. The biodistribution studies also showed the formation of a longlasting local depot following SC administration of the nanocapsules, a fact that would

NU

discourage the use of this administration route for the administration of DCX-loaded

MA

nanocapsules [38]. Overall, the observed access of PGA-PEG nanocapsules to the lymphatics upon IV administration, led us to assess their potential for the treatment of cancers associated with metastatic spreading in the lymph nodes. In this regard, we

TE

D

presumed that the enhanced permeability of the tumor-associated blood vessels could further promote the extravasation of nanocarriers to the tumor interstice and their

CE P

subsequent drainage by the peritumoral lymphatic vessels [15]. To study the performance of the PGA-PEG nanocapsules, we chose the taxane DCX as

AC

a reference drug. This selection was motivated not only by the well-known inherent toxicity of the taxanes-based marketed formulations, but also because of the possibility to compare the efficacy of PGA-PEG nanocapsules with the efficacy reported for other formulations containing paclitaxel (Abraxane®, Genexol-PM, Nanoxel-PM, Lipusu, NK-105 and Opaxio®) or DCX (BIND-014) already on the market, or currently under clinical development [39–42]. Regarding the systemic toxicity of DCX-loaded PGA-PEG nanocapsules, the results showed that their administration over 5 consecutive days, at a total accumulated dose of 60 mg/kg, does not trigger toxic effects. Indeed, the MTD for DCX-loaded nanocapsules was not achieved even when administered at doses higher than those 23

ACCEPTED MANUSCRIPT commonly used for therapeutic purposes in mice. On the contrary, the commercial formulation Taxotere® administered with the same dosing and schedule exhibited a

T

significant toxicity in the same time frame (Figure 2). This improved toxicological

IP

profile of the encapsulated drug agrees with the one previously reported by our group

SC R

for the anticancer drug plitidepsin [21]. In this previous study we could show a 3-fold increase in the single dose MTD of plitidepsin encapsulated in PGA-PEG nanocapsules compared to the standard formulation (i.e. ethanol/Cremophor EL/water vehicle). The

NU

reduced toxicity of the DCX nanocapsules, when compared with Taxotere®, could be attributed to an improved drug distribution within the organism. In addition, the toxicity

MA

of the marketed drug has also been attributed to the high concentration of Tween® 80 in the formulation [43]. These results are also in line with results reported in other works

TE

D

regarding the low toxicity of lipid nanocapsules [44] and nanoparticles [45–49] for the administration of taxanes.

CE P

With regard to the efficacy of DCX-loaded nanocapsules, it is worth mentioning that despite numerous reports on the efficacy of taxanes-loaded nanocarriers, only a few of

AC

them have specifically addressed their capacity to fight against metastasis. For example, micelles based on phosphatidylethanolamine, PGA-PEG or poly(lactide)-PEG, and polymeric or lipid-based nanoparticles have been shown to reduce metastasis in lymph nodes in orthotopic breast, gastric or melanoma cancer models [6–9,11–13]. However, whether this positive response, is due to a direct effect on the lymph nodes-resident cancer cells or, the indirect consequence of the size reduction of the primary tumor, is unclear. To evaluate the anti-metastatic effect, we have adapted an orthotopic lung metastasic model. This model reproduces the tumor environment and the cancer dissemination pathways in a more realistic way than the heterotopic subcutaneous implanted cancer 24

ACCEPTED MANUSCRIPT models classically used. The efficacy of DCX-loaded nanocapsules was evaluated after their administration at different times: at days 5 and 20 or 20 and 27 after the injection

T

of cancer cells. In the first case, the results indicate that the efficacy of DCX

IP

nanocapsules is similar to that of Taxotere®. This limited efficacy was attributed to the

SC R

fact that the treatments were applied before the spreading of cancer cells to the mediastinal lymph nodes (Figure 3). Thus, in this administration regime the therapeutic effect on the primary tumor can obscure a differential effect on the lymph node

NU

metastasis: the small number of cancer cells found in the lymphatic nodes after

MA

treatment could be an indirect consequence of the effect achieved over the primary tumor, i.e. the primary tumor has lost its capacity to initiate cell spreading, although it could also be due to a combined effect of the DCX formulations both in the primary

TE

D

tumor and in the lymph nodes. To answer to these hypotheses, we performed a second in vivo experiment, in which treatments started after the consolidation of lymphatic

CE P

metastasis in the mediastinal nodes (20 and 27 days after inoculation with A549luc cells in the lung). The results of this experiment led us to the conclusion that DCX

AC

nanocapsules (20 mg/kg) were active both, in the primary tumor and in the mediastinal nodes, whereas the commercial formulation was not (Figure 4 and 5). The explanation of a cytotoxic direct effect on the lymphatic nodes is consistent with the results of the biodistribution of the nanocapsules. The results of the studies presented here indicate that PGA-PEG nanocapsules were able to significantly decrease the tumor cell burden in the lungs (Figure 4 and 5) and the metastatic burden in the lymph nodes at day 47. Furthermore, a clear conclusion from these data is that the treatment with DCX-loaded nanocapsules was the only one capable of significantly reducing lymphatic metastases (Figure 5), and substantially improves the survival rate (Figure 4B). The enhanced antitumor efficacy of the nanocapsules 25

ACCEPTED MANUSCRIPT could be attributed to their higher accumulation in the tumor and draining lymph nodes, as well as to a prolonged DCX delivery from the nanocapsules. Another mechanism that

T

could be implicated in the superiority of DCX-loaded nanocapsules over Taxotere®

IP

could be their preferential internalization in cancer cells. This capacity to act on cancer

SC R

cells may represent an important advantage over other nanocarriers designed for the lymphatic targeting of drugs which, despite their significant uptake by the lymph nodes, have shown a limited anti-metastatic efficacy [19].

NU

Furthermore, the different outcomes observed for the two administration regimes of the

MA

treatment (before – Figure 3- and after –Figure 4- metastasis are developed) suggest that the nanoformulation would be particularly effective for the treatment of metastasis in an

CONCLUSIONS

TE

D

advanced cancer situation.

CE P

In conclusion, the results of this study show that small (100 nm) PGA-PEG nanocapsules are efficient carriers for the targeted delivery of DCX to both, the tumor

AC

tissue and the lymph nodes. This efficient delivery was translated into a drastic reduction of DCX systemic toxicity and an improvement of the efficacy in terms of tumor burden and metastatic spread. Therefore, we propose that the use of small PGAPEG nanocapsules may represent a promising approach for the treatment of metastatic lung cancer.

Acknowledgments The authors acknowledge financial support from the European Commission FP7 for the LYMPHOTARG (ERA-NET EuroNanoMed Program-ISCIII, ref PS09/02670) and NICHE projects (ERA-NET EuroNanoMed II framework by ISCIII through CIBER26

ACCEPTED MANUSCRIPT BBN) and Xunta de Galicia (Isidro Parga Pondal Fellowhsip and Competitive Reference Groups-FEDER Funds GRC2013-043). Abellan-Pose also acknowledges her

T

fellowship from the Doctoral School of Biomedical Sciences and Health Technologies -

IP

University of Santiago de Compostela. We also acknowledge the technical assistance of

SC R

Rafael Romero with the animals.

Conflict of interest

MA

NU

Authors declare no potential conflict of interest regarding the content of this work.

REFERENCES

J. Ferlay, I. Soerjomataram, M. Ervik, R. Dikshit, S. Eser, C. Mathers, et al., GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide, IARC CancerBase No. 11 [Internet]. Lyon, Fr. Int. Agency Res. Cancer. (2013). http://globocan.iarc.fr (accessed March 3, 2016).

[2]

R.L. Siegel, K.D. Miller, A. Jemal, Cancer Statistics, 2015, CA. Cancer J. Clin. 65 (2015) 5–29. doi:10.3322/caac.21254.

[3]

A. Schroeder, D.A. Heller, M.M. Winslow, J.E. Dahlman, G.W. Pratt, R. Langer, et al., Treating metastatic cancer with nanotechnology., Nat. Rev. Cancer. 12 (2012) 39–50. doi:10.1038/nrc3180.

[4]

S.A. Stacker, M.G. Achen, L. Jussila, M.E. Baldwin, K. Alitalo, Lymphangiogenesis and cancer metastasis., Nat. Rev. Cancer. 2 (2002) 573–83. doi:10.1038/nrc863.

[5]

R. Abellan-Pose, N. Csaba, M.J. Alonso, Lymphatic Targeting of Nanosystems for Anticancer Drug Therapy, Curr. Pharm. Des. 22 (2015) 1194–1209. doi:10.2174/1381612822666151216150809.

[6]

M. Rafi, H. Cabral, M.R. Kano, P. Mi, C. Iwata, M. Yashiro, et al., Polymeric micelles incorporating (1,2-diaminocyclohexane)platinum (II) suppress the growth of orthotopic scirrhous gastric tumors and their lymph node metastasis., J. Control. Release. 159 (2012) 189–96. doi:10.1016/j.jconrel.2012.01.038.

[7]

L. Qin, F. Zhang, X. Lu, X. Wei, J. Wang, X. Fang, et al., Polymeric micelles for enhanced lymphatic drug delivery to treat metastatic tumors., J. Control. Release. 171 (2013) 133–42. doi:10.1016/j.jconrel.2013.07.005.

[8]

H. Cabral, J. Makino, Y. Matsumoto, P. Mi, H. Wu, T. Nomoto, et al., Systemic Targeting of Lymph Node Metastasis through the Blood Vascular System by Using Size-Controlled Nanocarriers, ACS Nano. 9 (2015) 4957–4967. doi:10.1021/nn5070259.

AC

CE P

TE

D

[1]

27

ACCEPTED MANUSCRIPT [9]

Y. Li, M. Jin, S. Shao, W. Huang, F. Yang, W. Chen, et al., Small-sized polymeric micelles incorporating docetaxel suppress distant metastases in the clinically-relevant 4T1 mouse breast cancer model., BMC Cancer. 14 (2014) 329. doi:10.1186/1471-2407-14-329.

IP

T

[10] A. Estella-Hermoso de Mendoza, M.A. Campanero, H. Lana, J.A. Villa-Pulgarin, J. de la Iglesia-Vicente, F. Mollinedo, et al., Complete inhibition of extranodal dissemination of lymphoma by edelfosine-loaded lipid nanoparticles., Nanomedicine (Lond). 7 (2012) 679–90. doi:10.2217/nnm.11.134.

SC R

[11] R. Liu, D.M. Gilmore, K.A. V Zubris, X. Xu, P.J. Catalano, R.F. Padera, et al., Prevention of nodal metastases in breast cancer following the lymphatic migration of paclitaxel-loaded expansile nanoparticles., Biomaterials. 34 (2013) 1810–9. doi:10.1016/j.biomaterials.2012.11.038.

NU

[12] R. Tan, M. Niu, J. Zhao, Y. Liu, N. Feng, Preparation of vincristine sulfateloaded poly (butylcyanoacrylate) nanoparticles modified with pluronic F127 and evaluation of their lymphatic tissue targeting., J. Drug Target. 22 (2014) 509–17. doi:10.3109/1061186X.2014.897708.

MA

[13] B. Lu, S.-B. Xiong, H. Yang, X.-D. Yin, R.-B. Chao, Solid lipid nanoparticles of mitoxantrone for local injection against breast cancer and its lymph node metastases., Eur. J. Pharm. Sci. 28 (2006) 86–95. doi:10.1016/j.ejps.2006.01.001.

TE

D

[14] N. Wauthoz, G. Bastiat, E. Moysan, A. Cieslak, K. Kondo, M. Zandecki, et al., Safe lipid nanocapsule-based gel technology to target lymph nodes and combat mediastinal metastases from an orthotopic non-small-cell lung cancer model in SCID-CB17 mice, Nanomedicine Nanotechnology, Biol. Med. 1 (2015). doi:10.1016/j.nano.2015.02.010.

CE P

[15] G.M. Ryan, L.M. Kaminskas, C.J.H. Porter, Nano-chemotherapeutics: Maximising lymphatic drug exposure to improve the treatment of lymphmetastatic cancers., J. Control. Release. 193 (2014) 241–56. doi:10.1016/j.jconrel.2014.04.051.

AC

[16] D.A. Rao, M.L. Forrest, A.W.G. Alani, G.S. Kwon, J.R. Robinson, Biodegradable PLGA Based Nanoparticles for Sustained Regional Lymphatic Drug Delivery, J. Pharm. Sci. 99 (2010) 2018–2031. doi:10.1002/jps. [17] C.D. Kaur, M. Nahar, N.K. Jain, Lymphatic targeting of zidovudine using surface-engineered liposomes., J. Drug Target. 16 (2008) 798–805. doi:10.1080/10611860802475688. [18] S.M. Moghimi, The effect of methoxy-PEG chain length and molecular architecture on lymph node targeting of immuno-PEG liposomes., Biomaterials. 27 (2006) 136–44. doi:10.1016/j.biomaterials.2005.05.082. [19] L.M. Kaminskas, V.M. McLeod, G.M. Ryan, B.D. Kelly, J.M. Haynes, M. Williamson, et al., Pulmonary administration of a doxorubicin-conjugated dendrimer enhances drug exposure to lung metastases and improves cancer therapy., J. Control. Release. 183 (2014) 18–26. doi:10.1016/j.jconrel.2014.03.012. [20] M. Pitorre, G. Bastiat, E.M. dit Chatel, J.-P. Benoit, Passive and specific targeting of lymph nodes: the influence of the administration route, Eur. J. Nanomedicine. 7 (2015) 121–128. doi:10.1515/ejnm-2015-0003. 28

ACCEPTED MANUSCRIPT [21] T. Gonzalo, G. Lollo, M. Garcia-Fuentes, D. Torres, J. Correa, R. Riguera, et al., A new potential nano-oncological therapy based on polyamino acid nanocapsules., J. Control. Release. 169 (2013) 10–6. doi:10.1016/j.jconrel.2013.03.037.

IP

T

[22] G. Lollo, P. Hervella, P. Calvo, P. Avilés, M.J. Guillén, M. Garcia-Fuentes, et al., Enhanced in vivo therapeutic efficacy of plitidepsin-loaded nanocapsules decorated with a new poly-aminoacid-PEG derivative, Int. J. Pharm. 483 (2015) 212–219. doi:10.1016/j.ijpharm.2015.02.028.

SC R

[23] R. Abellan-Pose, C. Teijeiro-Valiño, M.J. Santander-Ortega, E. Borrajo, A. Vidal, M. Garcia-Fuentes, et al., Polyaminoacid Nanocapsules for Drug Delivery to the Lymphatic System: Effect of the Particle Size, Int. J. Pharm. 509 (2016) 107–117. doi:10.1016/j.ijpharm.2016.05.034.

NU

[24] S.H. Lee, S.D. Yoo, K.H. Lee, Rapid and sensitive determination of paclitaxel in mouse plasma by high-performance liquid chromatography., J. Chromatogr. Biomed. Sci. Appl. 724 (1999) 357–363. doi:10.1016/S0378-4347(98)00566-0.

MA

[25] Z.Y. Cui, J.S. Ahn, J.Y. Lee, W.S. Kim, Y.H. Lim, H.J. Jeon, et al., Mouse Orthotopic Lung Cancer Model Induced by PC14PE6, Cancer Res. Treat. 38 (2006) 234–239. doi:10.4143/crt.2006.38.4.234.

D

[26] Y. Liu, Y.-C. Tseng, L. Huang, Biodistribution studies of nanoparticles using fluorescence imaging: a qualitative or quantitative method?, Pharm. Res. 29 (2012) 3273–7. doi:10.1007/s11095-012-0818-1.

TE

[27] J. Mérian, J. Gravier, F. Navarro, I. Texier, Fluorescent nanoprobes dedicated to in vivo imaging: from preclinical validations to clinical translation., Molecules. 17 (2012) 5564–91. doi:10.3390/molecules17055564.

CE P

[28] C. Oussoren, J. Zuidema, D.J.A. Crommelin, G. Storm, Lymphatic uptake and biodistribution of liposomes after subcutaneous injection. II. Influence of liposomal size, lipid composition and lipid dose, Biochim. Biophys. Acta. (1997) 261–272. doi:10.1016/S0005-2736(97)00122-3.

AC

[29] U. Vanhoefer, S. Cao, A. Harstrick, S. Seeber, Y.M. Rustum, Comparative antitumor efficacy of docetaxel and paclitaxel in nude mice bearing human tumor xenografts that overexpress the multidrug resistance protein (MRP), Ann. Oncol. 8 (1997) 1221–1228. doi:10.1023/A:1008290406221. [30] M. V Lozano, D. Torrecilla, D. Torres, A. Vidal, F. Domínguez, M.J. Alonso, Highly efficient system to deliver taxanes into tumor cells: docetaxel-loaded chitosan oligomer colloidal carriers., Biomacromolecules. 9 (2008) 2186–93. doi:10.1021/bm800298u. [31] M. V Lozano, H. Esteban, J. Brea, M.I. Loza, D. Torres, M.J. Alonso, Intracellular delivery of docetaxel using freeze-dried polysaccharide nanocapsules., J. Microencapsul. 30 (2013) 181–188. doi:10.3109/02652048.2012.714411. [32] D. Torrecilla, M. V. Lozano, E. Lallana, J.I. Neissa, R. Novoa-Carballal, A. Vidal, et al., Anti-tumor efficacy of chitosan-g-poly(ethylene glycol) nanocapsules containing docetaxel: Anti-TMEFF-2 functionalized nanocapsules vs. non-functionalized nanocapsules, Eur. J. Pharm. Biopharm. 83 (2013) 330– 337. doi:10.1016/j.ejpb.2012.10.017. 29

ACCEPTED MANUSCRIPT [33] G.R. Rivera-Rodríguez, M.J. Alonso, D. Torres, Poly-L-asparagine nanocapsules as anticancer drug delivery vehicles., Eur. J. Pharm. Biopharm. 85 (2013) 481–7. doi:10.1016/j.ejpb.2013.08.001.

IP

T

[34] G.R. Rivera-Rodriguez, G. Lollo, T. Montier, J.P. Benoit, C. Passirani, M.J. Alonso, et al., In vivo evaluation of poly-l-asparagine nanocapsules as carriers for anti-cancer drug delivery., Int. J. Pharm. 458 (2013) 83–9. doi:10.1016/j.ijpharm.2013.09.038.

SC R

[35] F.A. Oyarzun-Ampuero, G.R. Rivera-Rodríguez, M.J. Alonso, D. Torres, Hyaluronan nanocapsules as a new vehicle for intracellular drug delivery., Eur. J. Pharm. Sci. 49 (2013) 483–90. doi:10.1016/j.ejps.2013.05.008.

NU

[36] G. Lollo, G.R. Rivera-Rodriguez, J. Bejaud, C. Passirani, J.P. Benoit, M. GarcíaFuentes, et al., Polyglutamic acid-PEG nanocapsules as long circulated carriers for the delivery of docetaxel, Eur. J. Pharm. Biopharm. 87 (2014) 47–54. doi:10.1016/j.ejpb.2014.02.004.

MA

[37] C. Oussoren, J. Zuidema, D. Croramelin, G. Storm, Lymphatic uptake and biodistribution of liposomes after subcutaneous injection I . Influence of the anatomical site of injection, J. Liposome Res. 7 (1997) 85–99. doi:10.3109/08982109709035487.

D

[38] C. Oussoren, G. Storm, Liposomes to target the lymphatics by subcutaneous administration., Adv. Drug Deliv. Rev. 50 (2001) 143–56. doi:10.1016/S0169409X(01)00154-5.

CE P

TE

[39] L.H. Reddy, D. Bazile, Drug delivery design for intravenous route with integrated physicochemistry, pharmacokinetics and pharmacodynamics: Illustration with the case of taxane therapeutics, Adv. Drug Deliv. Rev. 71 (2014) 34–57. doi:10.1016/j.addr.2013.10.007.

AC

[40] T. Hamaguchi, Y. Matsumura, M. Suzuki, K. Shimizu, R. Goda, I. Nakamura, et al., NK105, a paclitaxel-incorporating micellar nanoparticle formulation, can extend in vivo antitumour activity and reduce the neurotoxicity of paclitaxel., Br. J. Cancer. 92 (2005) 1240–1246. doi:10.1038/sj.bjc.6602479. [41] J.W. Singer, Paclitaxel poliglumex (XYOTAX, CT-2103): A macromolecular taxane, J. Control. Release. 109 (2005) 120–126. doi:10.1016/j.jconrel.2005.09.033. [42] J. Hrkach, D. Von Hoff, M. Mukkaram Ali, E. Andrianova, J. Auer, T. Campbell, et al., Preclinical development and clinical translation of a PSMA-targeted docetaxel nanoparticle with a differentiated pharmacological profile, Sci. Transl. Med. 4 (2012) 128ra39. doi:10.1126/scitranslmed.3003651. [43] Z. Weiszhár, J. Czúcz, C. Révész, L. Rosivall, J. Szebeni, Z. Rozsnyay, Complement activation by polyethoxylated pharmaceutical surfactants: Cremophor-EL, Tween-80 and Tween-20., Eur. J. Pharm. Sci. 45 (2012) 492–8. doi:10.1016/j.ejps.2011.09.016. [44] J. Hureaux, F. Lagarce, F. Gagnadoux, M.C. Rousselet, V. Moal, T. Urban, et al., Toxicological study and efficacy of blank and paclitaxel-loaded lipid nanocapsules after i.v. administration in mice, Pharm. Res. 27 (2010) 421–430. doi:10.1007/s11095-009-0024-y. [45] M.J. Ernsting, W.-L.

Tang,

N.W. MacCallum, S.-D.

Li,

Preclinical 30

ACCEPTED MANUSCRIPT pharmacokinetic, biodistribution, and anti-cancer efficacy studies of a docetaxelcarboxymethylcellulose nanoparticle in mouse models., Biomaterials. 33 (2012) 1445–54. doi:10.1016/j.biomaterials.2011.10.061.

IP

T

[46] H. Devalapally, D. Shenoy, S. Little, R. Langer, M. Amiji, Poly(ethylene oxide)modified poly(beta-amino ester) nanoparticles as a pH-sensitive system for tumor-targeted delivery of hydrophobic drugs: Part 3. Therapeutic efficacy and safety studies in ovarian cancer xenograft model, Cancer Chemother. Pharmacol. 59 (2007) 477–484. doi:10.1007/s00280-006-0287-5.

SC R

[47] L. Peng, A.N. Schorzman, P. Ma, A.J. Madden, W.C. Zamboni, S.R. Benhabbour, et al., 2′-(2-Bromohexadecanoyl)-Paclitaxel Conjugate Nanoparticles for the Treatment of Non-Small Cell Lung Cancer in an Orthotopic Xenograft Mouse Model, Int. J. Nanomedicine. 9 (2014) 3601–3610. doi:10.2147/IJN.S66040.

MA

NU

[48] I. V Zhigaltsev, G. Winters, M. Srinivasulu, J. Crawford, M. Wong, L. Amankwa, et al., Development of a weak-base docetaxel derivative that can be loaded into lipid nanoparticles., J. Control. Release. 144 (2010) 332–40. doi:10.1016/j.jconrel.2010.02.029.

AC

CE P

TE

D

[49] Z. Xu, L. Chen, W. Gu, Y. Gao, L. Lin, Z. Zhang, et al., The performance of docetaxel-loaded solid lipid nanoparticles targeted to hepatocellular carcinoma., Biomaterials. 30 (2009) 226–32. doi:10.1016/j.biomaterials.2008.09.014.

31

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Graphical abstract

32