Improved pharmacokinetic profile of lipophilic anti-cancer drugs using ανβ3-targeted polyurethane-polyurea nanoparticles

    Improved Pharmacokinetic Profile of Lipophilic Anti-Cancer Drugs Using ανβ3-targeted Polyurethane-Polyurea Nanoparticles Pau Rocas PhD, Yolanda Fern´andez PhD, Natalia Garc´ıa-Aranda, Laia Foradada PhD, Pilar Calvo PhD, Pablo Avil´es PhD, Mar´ıa Jos´e Guill´en PhD, Sim´o Schwartz Jr. MD, PhD, Josep Rocas PhD, Fernando Albericio PhD Prof, Ibane Abasolo PhD PII: DOI: Reference:

S1549-9634(17)30189-2 doi: 10.1016/j.nano.2017.10.009 NANO 1679

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

11 April 2017 4 September 2017 20 October 2017

Please cite this article as: Rocas Pau, Fern´andez Yolanda, Garc´ıa-Aranda Natalia, Foradada Laia, Calvo Pilar, Avil´es Pablo, Guill´en Mar´ıa Jos´e, Schwartz Jr. Sim´o, Rocas Josep, Albericio Fernando, Abasolo Ibane, Improved Pharmacokinetic Profile of Lipophilic Anti-Cancer Drugs Using ανβ3-targeted Polyurethane-Polyurea Nanoparticles, Nanomedicine: Nanotechnology, Biology, and Medicine (2017), doi: 10.1016/j.nano.2017.10.009

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ACCEPTED MANUSCRIPT Improved Pharmacokinetic Profile of Lipophilic Anti-Cancer Drugs Using ανβ3-targeted Polyurethane-Polyurea Nanoparticles

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Pau Rocas, PhDa,b,1, Yolanda Fernández, PhD c,d,1, Natalia García-Aranda c,d, Laia Foradada, PhD c,d2, Pilar Calvo, PhD e, Pablo Avilés, PhD e, María José

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Guillén, PhD e, Simó Schwartz Jr., MD PhD c,d, Josep Rocas, PhD b*, Fernando Albericio, PhD Prof. a,d,f,g*, Ibane Abasolo, PhD c,d* Institute for Research in Biomedicine (IRB Barcelona), Baldiri Reixac 10, 08028 Barcelona,

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a

Spain b

Nanobiotechnological Polymers Division, Ecopol Tech S.L., Indústria 7, 43720 L'Arboç, Spain

c

Functional Validation & Preclinical Research (FVPR), Drug Delivery and Targeting Group,

CIBBIM-Nanomedicine, Vall d'Hebron Institut de Recerca (VHIR), Universitat Autònoma de Barcelona (UAB), 08035 Barcelona, Spain

Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-

BBN), Barcelona, Spain

PharmaMar S.A., Avda de los Reyes, 1 Pol. Ind. La Mina, 28770 Colmenar Viejo, Madrid,

Spain f

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e

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d

Department of Organic Chemistry, University of Barcelona, Martí Franquès 1-11, 08028

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Barcelona, Spain

School of Chemistry & Physics, University of Kwazulu-Natal, 4041 Durban, South Africa

1

Both authors contributed equally to this work, PR performed the synthesis and

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g

physicochemical characterization of the nanoparticles while YF was in charge of in vitro and in vivo assays. 2

Current address: Vall d’Hebron Institute of Oncology (VHIO), 08035 Barcelona, Spain

*Corresponding authors: J.R., F.A., I.A *Main corresponding author: I.A. Functional Validation & Preclinical Research (FVPR), Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain E-Mail: [email protected]

Phone: +34 93-489-3000 ext. 3375

Funding: This work was supported by MICINN and FEDER [IPT-090000-2010-0001, POLYSFERA project], CICYT [CTQ2015-67870-P], Generalitat de Catalunya [2014 SGR 137],

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ACCEPTED MANUSCRIPT and “Fundació Marató TV3” [337/C/2013]. The Spanish Ministry of Science and Innovation also

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supported NG-A as laboratory technician [PTA2013-8431-I].

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ABSTRACT WORD COUNT: 150

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TEXT WORD COUNT: 4,973 NUMBER OF REFERENCES: 39

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NUMBER OF FIGURES: 8

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ACCEPTED MANUSCRIPT ABSTRACT Glutathione degradable polyurethane-polyurea nanoparticles (PUUa NP) with a disulfide-rich multiwalled structure and a cyclic RGD peptide as a targeting moiety were synthesized,

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incorporating a very lipophilic chemotherapeutic drug named Plitidepsin. In vitro studies indicated that encapsulated drug maintained and even improved its cytotoxic activity while in vivo toxicity studies revealed that the maximum tolerated dose (MTD) of Plitidepsin could be

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increased three-fold after encapsulation. We also found that pharmacokinetic parameters such as maximum concentration (Cmax), area under the curve (AUC) and plasma half-life were

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significantly improved for Plitidepsin loaded in PUUa NP. Moreover, biodistribution assays in mice showed that RGD-decorated PUUa NP accumulate less in spleen and liver than nontargeted conjugates, suggesting that RGD-decorated nanoparticles avoid sequestration by macrophages from the reticuloendothelial system. Overall, our results indicate that

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polyurethane-polyurea nanoparticles represent a very valuable nanoplatform for the delivery of lipophilic drugs by improving their toxicological, pharmacokinetic and whole-body biodistribution

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KEYWORDS

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profiles.

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Polyurethane-polyurea nanoparticles, RGD peptide, Plitidepsin, cancer, biodistribution

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ACCEPTED MANUSCRIPT BACKGROUND Most treatment regimes for fighting cancer are based on water insoluble chemotherapeutic 1

agents such etoposide, paclitaxel or camptothecin . This characteristic does not only difficult

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the formulation of the active drugs but also burdens its in vivo efficacy. Poorly soluble lipophilic drugs often show low bioavailability, do not distribute properly in the body and do not effectively 2

accumulate within the tumors . Consequently, higher doses of drugs are needed and side-

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effects and mechanisms of drug resistance appear. Encapsulation of poorly water-soluble molecules in nanoparticles has been seen as a way to overcome these problems. Although

the market

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different types of nanomedicines incorporating chemotherapeutic drugs have already reached there is still a strong demand to develop alternative delivery vehicles with which to

improve the efficacy/toxicity profiles of lipophilic drugs.

In this regard, investigators have put efforts to create multilayered nano-microstructures in order

active principle to the carrier

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to enhance the encapsulation and stability of drugs without the need of covalently linking the 4,5

. These systems could be decorated with polyethylene glycol

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which can reduce opsonization and phagocytosis from the

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(PEG) or zwitteronic coatings

reticulo-endothelial system (RES). Because nanoparticles are not efficiently scavenged by

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macrophages, increased blood circulation times and bioavailability are expected to extend the duration of controlled drug delivery and to improve the prospects of nanoparticles to reach 7

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target sites by extravasation . This is especially relevant for targeting solid tumors, where extension in circulation time is combined with the known phenomena of enhanced permeability and retention (EPR)

8,9

. Moreover, the presence of specific targeting moieties might change the

biodistribution profile of the nanoparticle by increasing its presence at tumor sites

10

or it might

improve the final efficacy of the system by facilitating the internalization of the drug in cancer cells

11,12

. In this sense, nanosystems functionalized with specific arginine-glycine-aspartic acid

(RGD)-containing peptides binding integrin αvβ3, have shown not only to increase integrin affinity and clustering but also to induce active integrin-mediated internalization

13–15

.

Here we report on a multiwalled drug delivery system based in polyurethane-polyurea nanoparticles (PUUa NP) able to improve encapsulation stability of poorly water-soluble drugs, and to enhance their pharmacokinetic parameters in vivo. Nanoparticles with a liquid oily core

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ACCEPTED MANUSCRIPT and a crosslinked shell combining two prepolymers (one hydrophobic and another amphiphilic) and a hydrophilic RGD peptide were efficiently developed following procedures we recently described

16–19

. PUUa NP offer specific advantages as drug delivery systems for anti-cancer

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drugs: possibility of high range of chemical modulation of polymer shell thanks to the high

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reactivity of isocyanates with amino and hydroxyl groups, very stable and efficient encapsulation of drugs thanks to the self-stratified and crosslinked PUUa NP shell and easy formulation

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(lyophilizable without cryoprotectants and fast re-dispersing capacity in aqueous media). As a drug model, we used Plitidepsin a marine origin drug currently in Phase III of clinical stage 20

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for myeloma (NCT02100657) and lymphoma (NCT03070964)

. Originally isolated from the

Mediterranean tunicate Aplidium albicans, Plitidepsin is currently produced by chemical synthesis. This is a highly hydrophobic drug, and although Plitidepsin has shown strong anticancer activity against a large variety of cultured human cancer cells

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, its poor

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biodistribution and bioavailability renders a narrow therapeutic window. With the aim of improving the efficacy/toxicity balance of Plitidepsin, we synthesized and characterized

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Plitidepsin-loaded PUUa NP superficially functionalized with cyclic RGD peptides. This peptide selectively binds integrin αvβ3 overexpressed on activated endothelial cells of growing vessels

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and on tumor cells. Following extensive in vitro characterization of PUUa NP, we assessed the in vivo proof-of-principle for their capacity to improve the maximum tolerated dose (MTD), the

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pharmacokinetic parameters and the whole-body biodistribution of the nanoencapsulated drug ®

compared to the reference formulation (Cremophor EL solution).

METHODS 1. Chemicals Plitidepsin was kindly provided by PharmaMar S.A. (Madrid, Spain). Cyclo(-Arg-Gly-Asp-D-PheLys) (cRGDfK) was synthesized according to previously reported protocols

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. YMER™ N-120

was obtained as a free sample from Perstorp (Perstorp, Sweden), the same as N-dodecyl-1,3propylenediamine (LAP 100D) by Clariant (Barcelona, Spain). Capric/caprylic triglyceride mixture (Crodamol GTCC) was obtained from Croda (Barcelona, Spain) and Bayhydur 3100 was purchased from Bayer (Leverkusen, Germany). If not indicated otherwise, all other

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ACCEPTED MANUSCRIPT reagents were purchased from Sigma-Aldrich (St Louis, MO, USA). Extra dry acetone was used during all the synthetic process. 2. Synthesis and characterization of reactive prepolymers and polyurethane-

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polyurea nanoparticles (PUUa NP)

Preparation of the reactive amphiphilic prepolymer (Amphil) and the Bayhydur 3100-cRGDfK 18

. The

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conjugate (B3100-cRGDfK) followed previously described methods with slight changes

formation of polyurethane and polyurethane-polyurea prepolymers was followed by FTIR and 17,18,23

while the presence of one cRGDfK molecule per linker and total

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characterized by NMR

concentration cRGDfK was ensured by MALDI-TOF MS and HPLC, respectively

17,18

.

To obtain PUUa NP, previously homogenized Amphil+IPDI (1.73 g, mass ratio Amphil 10.6:1 IPDI) was added to a round-bottom flask containing B3100 (125 mg, 0.167 mmol) or B3100-

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cRGDfK (16% w/w) and DiR (1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanine iodide) fluorochrome (5 mg, 5 µmol) and/or Plitidepsin under nitrogen atmosphere. This organic mixture

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was then emulsified in PBS (16 mL, pH 7.4, 5ºC) with a magnetic stirrer in an ice bath to prevent isocyanate reaction with water. Once emulsified, diethylenetriamine (DETA) (76 mg,

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0.73 mmol) was added and crosslinked nanoparticles were formed by interfacial polyaddition as proved by FTIR. Acetone was removed in a rotavapor. PUUa NP were dialyzed (100000

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MWCO, Spectrum Laboratories, California, USA) against pure water or PBS during 72 h. Physicochemical characterization of PUUa NP included transmission electronic microscopy (TEM), Dynamic Light Scattering (DLS) and HPLC quantification as detailed in Supplementary Information (Supporting Methods)

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. Previously dialyzed formulations at 10% solids (w/w) were

lyophilized, directly redispersed at desired concentration and examined by TEM and DLS to ratify optimal size and morphology characteristics (see Supporting Information). Thereafter, every experiment shown in the main text was performed with lyophilized and redispersed PUUa NP either in pure milliQ grade water or PBS. Alternative information can be found in the Supporting Information or previous publications

17,18,24

.

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ACCEPTED MANUSCRIPT 3. Plitidepsin and DiR encapsulation and release To quantify the total amount of encapsulated Plitidepsin in PUUa NP, a calibration curve was performed by preparing standard solutions of Plitidepsin in CH3CN:H2O (1:1, v/v) for HPLC

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analysis. Lyophilized PUUa NP loaded with Plitidepsin (5 mg) were emulsified in 2 mL CH3CN:DMSO (95:5, v/v) during one week at 37ºC to extract the drug and placed in a centrifugal 3 KDa filter unit (Microcon, Carrigtwohill, Ireland) and centrifuged at 14,000 g for 30

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min. Analytical HPLC runs of the filtrate were appropriately diluted in CH3CN:H2O (1:1 v/v) and analyzed in triplicate in a Waters 2998 HPLC using a X-Bridge BEH130, C18, 3.5 µm, 4.6 X

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100-mm reverse-phase column with the following gradient: 50% to 100% of B in 8 min at a flow rate of 1 mL/min; eluent A: H2O with 0.045% TFA (v/v); eluent B: CH3CN with 0.036% TFA (v/v) and UV detection at 225 nm.

In vitro drug release from nanoparticles was evaluated either in a control buffer composed by

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PBS (0.01 M) with 4% (w/w) bovine serum albumin (BSA) and 1% (v/v) Tween 20 or in a nanoparticle degradation buffer formed by the same components plus 10 mM glutathione

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(GSH). The concentration of Plitidepsin in the medium was studied by ultrafiltration, measuring the difference between the filtrated and the encapsulated Plitidepsin. The filtrate was

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appropriately diluted in CH3CN:H2O 1:1 (v/v) and analyzed at different time intervals following

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the method described above. 4. In vitro efficacy studies Based on the potential indications for Plitidepsin, cell lines derived from human glioblastoma (U87-MG), colorectal cancer (HT-29 and HCT 116) and breast cancer (MDA-MB-231) were used in cytotoxicity assays all expressing different levels of αvβ3 and αvβ5 integrins

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. All cells

were obtained from the American Type Culture Collection and maintained in a humid atmosphere at 37°C with 5% CO2. Briefly, U87-MG cells were cultured in DMEM medium, HT29 and HCT 116 cells in RPMI 1640 and MDA-MB-231 in DMEM/F12 medium (Life Technologies, Madrid, Spain), all supplemented with 10% fetal bovine serum. In vitro cytotoxicity of nanoparticles was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method after 72 h incubation following procedures previously described

18,25,26

or

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ACCEPTED MANUSCRIPT by incubating cells for 2 h with the nanoparticles, washing cells with PBS and letting them recover for additional 24 h.

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5. Toxicological evaluation

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The acute toxicity studies of Plitidepsin formulated in PUUa NP-DiR and PUUa NP-RGD-DiR were performed in CD-1 female (n=5 per group) after single intravenous (i.v.) administration of

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different doses of Plitidepsin (0.5 – 1.2 mg/kg). Toxicity of non-loaded PUUa NP was also examined at a maximum dose of 1 g/kg. In all cases, body weight, clinical signs and mortality

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were recorded daily during the whole assay (14 days). Any mouse showing signs of extreme weakness, toxicity or in a moribund state was euthanized following humanitarian endpoint criteria. The Maximum Tolerated Dose (MTD) was defined as the dose level resulting in less of 15% of weight loss and without any mortality record. All procedures for animal handling, care and treatment were carried out according the procedures approved by the regional Institutional

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Animal Care and Use Committees.

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6. Pharmacokinetic parameters evaluation The pharmacokinetic studies were performed in CD-1 female mice (n=54) upon a single i.v.

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administration of 2 different formulations (integrin targeted PUUa NP-DiR-PLI-RGD and nontargeted PUUa NP-DiR-PLI) at 0.20 mg/kg of Plitidepsin. Determination of drug content in

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plasma and tissue samples is described in the Supplementary information. Pharmacokinetic parameters of Plitidepsin were performed using a non-compartmental pharmacokinetic method a WinNonlin™ Professional Version 4.01 (Pharsight Corporation, Mountain View, CA, USA). The AUC values given were normalized to the dose given. . 7. In vivo biodistribution of PUUa NP-DiR All the in vivo biodistribution studies were performed by the ICTS “NANBIOSIS”, more specifically by the CIBER-BBN’s In Vivo Experimental Platform of the Functional Validation & Preclinical

Research

(FVPR)

area

(http://www.nanbiosis.es/unit/u20-in-vivo-

experimentalplatform/) (Barcelona, Spain). Briefly, 6 week-old female athymic nude mice (n=40)

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ACCEPTED MANUSCRIPT were intravenously administered with a single dose of integrin targeted (PUUa NP-DiR-PLIRGD) and non-targeted (PUUa NP-DiR-PLI) nanoparticles at 1.7 mg/kg of Plitidepsin (0.4 mg/kg of DiR) or the empty integrin targeted (PUUa NP-DiR-RGD) and non-targeted (PUUa NP-DiR)

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nanoparticles (0.4 and 1.5 mg/kg of DiR) resuspended in saline. Since observation time was

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shortened to 72 h and to ensure the observation of DiR fluorescence in vivo, the administered dose doubled the MTD found in toxicity assays. Thereafter, at 4, 6, 24, 48 and 72 h post

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administration non-invasive DiR fluorescent images were acquired using the IVIS Spectrum imaging system with the Living Image 4.3 software (PerkinElmer, MA, USA)

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. Mice were

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euthanized at 24 and 72 h post administration. Blood and major organs such as liver, spleen, kidneys and lungs were also imaged and then stored at −80°C to quantify Plitidepsin levels. Fluorescence image (FLI) quantification was performed over regions of interest (ROI) as Radiant Efficiency or Radiant Efficiency per tissue weight, once the DiR light emission in each

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compound was corrected.

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RESULTS

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Preparation and characterization of PUUa NP Novel PUUa NP were synthesized from a first step of polyaddition reaction followed by

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emulsification in water. Afterwards, crosslinking of pre-emulsified reactive monomers by interfacial polyaddition reaction was performed by adding a polyamine. The resulting PUUa NP (Figure 1) encompassed disulfide bonds in their crosslinked shell (Supplementary Figure S1 and S2), allowing a controlled intracellular release upon contact with cytosolic GSH, while the oily core incorporated the drug (Plitidepsin) and/or the near infrared dye (DiR) for in vivo monitoring purposes. In addition, PUUa NP were superficially decorated with the cRGDfK targeting peptide.

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Figure 1. PUUa NP synthetic process. Amphiphilic and hydrophobic prepolymers, lipophilic cargoes (drugs or lipophilic fluorophores) and cRGDfk targeting peptide are self-stratified in water by hydrophobic effects. After fixing the shell by interfacial crosslinking, the hydrophilic RGD targeting peptide is located at the surface of the PUUa NP allowing

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specific cell binding.

Robust multiwalled nanoparticles were fully characterized in terms of drug loading, morphology, size distribution and surface charge. To ensure high encapsulation efficiency (99%), Plitidepsin was loaded into the polymer nanoparticles with at a final drug loading of 1% (w/w) (0.9 mg/mL

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of Plitidepsin). DiR lipophilic fluorophore was encapsulated at a loading of 0.28% (w/w) (0.25 mg/mL). Regarding size, PUUa NP had a polymeric dense morphology with monodisperse sizes

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around 90-110 nm with Z-potential values ranging from 0 to 1. Moreover, these values did not changed when PUUa NP were loaded with Plitidepsin and DiR or functionalized with cRGDfK,

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cyclic RGD peptide (Figure 2, Table 1 and Figure S3).

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Moreover, PUUa NP were characterized by TEM, DLS and Z-potential before and after lyophilization showing that shape, size and surface charge of the nanoparticles did not change significantly after redispersation (Supplementary data, Table S1 and Figure S4).

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B

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Figure. 2. TEM micrographs of PUUa NP. A) PUUa NP-DiR; B) PUUa NP-DiR-PLI; c) PUUa NP-DiR-RGD; and C) PUUa NP-DiR-PLli-RGD. All nanoparticles showed a round spherical shape with very homogeneous sizes around 100

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nm.

Table 1. Physicochemical characterization of PUUa NP. Characterization of size by TEM and DLS, polidispersity

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index (PDI) of each measurement and the Z-potential of different PUUa NP loaded with DiR (0.25 mg/mL) and

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Nanoparticle

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Plitidepsin (0.9 mg/mL). Mean ± SD are indicated with an n=3 in all cases.

TEM

Diameter (nm)

DLS PDI

Diameter (nm)

Z-potential PDI

(mV)

PUUa NP DiR

111 ± 26

0.06

105 ± 37

0.13

0.90 ± 0.15

PUUa NP DiR-RGD

105 ± 31

0.09

106 ± 38

0.13

0.45 ± 0.10

PUUa NP DiR-PLI

108 ± 30

0.08

96 ± 33

0.11

0.38 ± 0.20

PUUa NP DiR-PLI-RGD

89 ± 37

0.17

91 ± 33

0.13

0.66 ± 0.10

Drug release studies Many cancer types are associated with elevated intracellular concentrations of GSH, a tripeptide with reductive potential involved in proliferative responses. PUUa NP developed here contain disulfide moieties in the polymeric shell to allow the drug release by intracellular levels 11

ACCEPTED MANUSCRIPT of GSH. To demonstrate this fact, the degradation by GSH and the changes on nanoparticles morphology were studied during one week. As seen from TEM micrographs (Figure 3), PUUa NP were gradually degraded and polymeric aggregates clearly appeared after one week of

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incubation in 10 mM GSH at 37ºC.

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Figure 3. Degradation of PUUa NP. TEM micrograph proving the nanoparticle degradation process over time in the presence (A, B, C) or absence (D,E,F) of disulphide bonds in the polymeric shell. While disulfide-bearing NPs change

conditions.

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their morphology over time in the presence of GSH, their disulfide-free counterparts remain stable under the same

Drug release experiments confirmed that PUUa NP were very stable and only released the drug in presence of GSH. When PUUa NP-PLI were incubated with PBS-BSA-Tween20 (control media) minimal release was observed over 144 h. However, when PUUa NP-PLI were incubated with PBS-BSA-Twen20-GSH (degradation media) a quite rapid released was observed for the first 10 h (~30%) that followed with a slower release rate that was sustained at 144 h (70%) (Figure 4). In addition, inexistent release of DiR dye after 24 h incubation of PUUa NP-DiR in PBS containing BSA (4%, w/w) and Tween 20 (1%, w/w) was observed (results not shown), which made them particularly appropriated for the in vivo fluorescence monitoring.

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100 80 Degradation media Control media

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40 20 0 0

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Time (h)

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(Mean  SD)

Plitidepsin Release (%)

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Figure 4. Cumulative Plitidepsin drug release from PUUa NP. Plitidepsin containing PUUa NP were incubated in degradation media (10 mM GSH, squares) or control media (no GSH, circles) and Plitidepsin drug concentrations were

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determined by HPLC. Mean ± SD are indicated with an n=2 in all cases.

In vitro efficacy

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To further explore drug-release and efficacy of encapsulated Plitidepsin in a cellular environment, cancer cell lines with different levels of integrin αvβ3 expression (Supplementary

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data, Table S2) were incubated with increasing concentrations of PUUa NP. Cell cytotoxicity assays clearly showed that Plitidepsin loaded PUUa NP were as effective as the free drug

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(Figure 5A and Figure 5C and Table 2) when incubated continuously for 72 h. At shorter incubation times the presence of the RGD moiety revealed to increase the efficacy of the PUUa

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NP, as shown for U87-MG and MDA-MB-231 cells (Figure 5B and Figure 5D). In all studied cell lines, IC50 values of encapsulated Plitidepsin at 72 h were similar to those of free Plitidepsin, with the sole exception of MDA-MB-231. In this breast cancer cell line, known to be resistant to many chemotherapeutic agents, the efficacy of the PUUa NP encapsulated drug was improved (IC50 of drug loaded and targeted NP was 9.7 ± 2.7 nM) compared to free drug (IC50 value of 22.1 ± 7.4 nM).

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ACCEPTED MANUSCRIPT Table 2. IC50 values of Plitidepsin-loaded PUUa NP. Plitidepsin concentration (nM) corresponding to the mean IC50 value ± SEM in of 3 independent experiments, as determined in MTT assays.

PUUa NP-PLI-RGD

PUUa NP-PLI

PLI

U87-MG

1.258 ± 0.378

2.069 ± 0.927

2.261 ± 1.265

MDA-MB-231

9.684 ± 2.688

8.980 ±1.217

22.10 ± 7.357

HCT 116

1.117 ± 0.639

1.216 ± 0.586

0.616 ± 0.473

HT-29

2.304 ± 1.206

3.288 ± 1.663

0.904 ± 0.289

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Cell line

Figure. 5 In vitro efficacy of Plitidepsin-loaded PUUa NP in U87-MG and MDA-MB-231 cancer cell lines. MTT assays were performed after a long and continuous incubation during 72 h with PUUa (A and C) or for short-term 2 h incubation (B and D). To ensure the non-toxicity of the delivery system, non-loaded PUUa NP or non-conjugated

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ACCEPTED MANUSCRIPT cRGDfK PUUa NP were also included in the long-term MTT assays. Asterisks in B and D indicate significant or very significant differences between non-targeted and targeted nanoparticles after 2 h incubation (** for p<0.01 and *** for p<0.001). Data correspond to the mean of 3 independent experiments.

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Toxicological evaluation

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The in vivo toxicity of Plitidepsin-loaded PUUa NP was evaluated by determining the MTD in CD-1 mice. Previous studies had already established that the MTD of the reference formulation 27

. In our case, the

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of Plitidepsin (Cremophor® EL/ethanol/water 15/15/70 w/w/w) was 0.3 mg/kg

MTD of Plitidepsin-loaded PUUa NP in CD-1 mice was 0.9 mg/kg for both, targeted and non-

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targeted PUUa NP. Accordingly, encapsulation of Plitidepsin in PUUa NP reduces significantly the toxicity associated to the administration of Plitidepsin, widening the therapeutic window of the drug. Moreover, there were no differences in the maximum toxicity of targeted and nontargeted PUUa NP, indicating that the addition of the RGD moiety does not alter significantly the

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toxicological profile of the PUUa NP. Pharmacokinetic profile

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The main goal of encapsulating Plitidepsin into PUUa NP was to improve Plitidepsin’s plasma

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half time and its biodistribution. In order to assess the plasma half-life of the encapsulated drug in PUUa NP, Plitidepsin reference formulation, as well as RGD-targeted and non-targeted

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Plitidepsin-loaded PUUa NP, were administered intravenously to healthy CD-1 mice. The plasmatic drug concentrations following i.v. administration of the different formulations are shown in Figure 6.

(Mean  SD)

[Pli] (ng/mL)

1000 100

PUUa NP-RGD-Pli PUUa NP-Pli Pli

10 1 0.1 0

10

20

30

Time (h)

Figure. 6. Pharmacokinetic profile of Plitidepsin in PUUa NP. Plasma concentration of Plitidepsin was determined after i.v. administration of 2 mg/kg Plitidepsin PUUa NP to CD-1 mice. Each point represents the mean and SD of n = 3.

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ACCEPTED MANUSCRIPT Accordingly, Plitidepsin plasmatic levels achieved after administration of drug-loaded targeted and non-targeted PUUa NP were much higher than those corresponding to the free Plitidepsin. Similar conclusions can be drawn from Table 3, which shows the pharmacokinetic parameters

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associated to all formulations. Both RGD-targeted and non-targeted PUUa NP showed higher

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half-life and lower clearance rates compared to free Plitidepsin. All pharmacokinetic parameters analyzed indicated that the encapsulation of a lipophilic drug such Plitidepsin in PUUa NP might

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significantly improve the efficacy of the carried drug.

Table 3. Pharmacokinetic parameters of Plitidepsin-loaded PUUa NP after single i.v. administration to CD-1

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mice. Maximum plasma concentration (Cmax), Area under the curve (AUC), plasma half-life (t1/2), clearance (CLp) and volume of distribution (Vdss) were calculated from average plasma concentrations (n= 3).

Nanoparticle

Cmax

AUC0-24

t1/2

(ng.h/mL)

(h)

(mL/h/kg)

(mL/kg)

1212.8

11.7

0.0001

0.0018

506.0

1871.9

8.9

0.0001

0.0009

2.6

11.4

8.5

15657.9

137035.5

(ng/mL) 434.3

PUUa NP-DiR-PLI-RGD

Vdss

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Plitidepsin

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PUUa NP-DiR-PLI

CLp

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In vivo biodistribution

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In vivo monitoring of DiR-labeled PUUa NP biodistribution was feasible using DiR at a concentration of 1.25 and 0.65 mg DiR/kg (see Supplementary Data Fig S5). Non-invasive in vivo FLI showed that targeted RGD-decorated PUUa NP-DiR tended to accumulate less in liver that the non-targeted PUUa NP-DiR (see Figure 7A). Liver accumulation ratio increased up to 48 h post administration, and then after, the fluorescent signal tended to diminish for up to 72 h post administration. To further confirm the results obtained by in vivo FLI at 24 and 72 h post administration, a subgroup of mice were euthanized and livers were collected. As expected, ex vivo results corroborated what observed in vivo; therefore, targeted PUUa NP-DiR-RGD were less uptaken by the liver than the non-targeted PUUa NP-DiR (Figure 7B). Moreover, the liver accumulation slightly decreased from 24 to 72 h, indicating that the nanoparticles were at least partially metabolized and cleared through the liver.

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Figure 7. Liver accumulation of DiR-labelled PUUa NP in mice athymic mice. A) Non-invasive in vivo FLI images of

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integrin-targeted (PUUa NP-DiR-RGD) and non-targeted (PUUa NP-DiR-RGD) tissue biodistribution over time and B) semiquantitative estimation of fluorescence DiR intensity ratio in the liver area in vivo in respect to 1 h post administration. C) Ex vivo FLI liver images and D) quantification of liver DiR fluorescence per weight of tissue at 24 and

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72 h end time points. Pseudocolor scale bars were consistent for all images in order to show relative changes with time.

In order to better study the biodistribution of the Plitidepsin loaded PUUa NP, NP loaded with 1.7 mg/kg of Plitidepsin and labeled with 0.4 mg/kg of DiR fluorophore were also administered. Although administration of such dose of Plitidepsin caused a significant weight loss (T/C body weight ratio were -13% and -21% for the PUUa NP-DiR-PLI-RGD and PUUa NP-PLI-DiR, respectively, 3 days postadministration) such weight loss was manageable within the first 72 h and biodistribution assays could be completed. In these animals, a semi-quantitative estimation of fluorescence DiR intensity in plasma, liver, spleen, lung, kidney, heart, muscle and skin was performed by ex vivo FLI at 24 and 72 h post administration. Results showed that PUUa NP greatly biodistribute into liver, spleen and lungs (Figure 8). In agreement with previous results, this experiment confirmed that RGD-decorated PUUa NP accumulate less in liver and spleen

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(

% of

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10 0

Plasma

Liver

Spleen

Lung

Kidney

Heart

Muscle

Skin

than non-targeted PUUa NP, indicating that functionalization of nanoparticles reduces their PUUa NP-DiR-Pli_1.7 mg Pli/kg_24 h

PUUa NP-DiR-Pli_1.7 mg Pli/kg_72 h

PUUa In NP-DiR-Pli-RGD_1.7 Pli/kg_24 h no significant PUUa NP-DiR-Pli-RGD_1.7 Pli/kg_72 h sequestration by RES. the case ofmgplasma, differences weremgobserved between

RGD-targeted and non-targeted PUUa NP levels at 72 h post administration. As for the other tissues, similar tissue accumulation patterns were also observed in kidneys, heart, muscle and

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(Mean  SEM)

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% of DiR Intensity/g

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skin.

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Kidney

Heart

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PUUa NP-DiR-Pli-RGD_1.7 mg Pli/kg

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PUUa NP-DiR-Pli_1.7 mg Pli/kg

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Figure 8. Tissue biodistribution of integrin targeted and non-targeted PUUa NP loaded with Plitidepsin in

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athymic mice. Ex vivo plasma and tissue intensity per tissue weight at 72 h post administration of integrin targeted PUUa NP-DiR-PLI-RGD and non-targeted PUUa NP DiR-PLI at 1.7 mg PLI/kg and 0.4 mg DiR/kg by i.v. administration

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route is shown. Mean percentage values of each corresponding formulation tissue accumulation per tissue weight in respect to the total maximal tissue fluorescence are displayed in the graph. Data in the inserted table correspond to the mean ± standard deviation (SD) of Plitidepsin content measured by HPLC.

DiR biodistribution results were further corroborated by the quantification of Plitidepsin levels in tissues collected 24 h post administration of a single dose of DiR labeled PUUa NP. Liver and spleen tissues accumulated higher levels of drug when Plitidepsin (1.7 mg/kg) was delivered in non-targeted PUUa NP (4.32 ± 0.35 µg/g and 34.1 ± 15.7 µg/g for liver and spleen, respectively), whereas these tissues showed lower levels of drug when RGD-decorated PUUa NP were administered (3.68 ± 0.33 µg/g and 22.1 ± 8.1 µg/g for liver and spleen, respectively). Moreover, histopathological analysis of lever and spleen did not reveal any histological

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ACCEPTED MANUSCRIPT alterations in these organs, precluding any toxic effect of PUUa NP at the administered doses (Figure 6S).

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DISCUSION

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Herein we report the use of PUUa NP for improving the biodistribution and pharmacokinetic profile of a highly insoluble drug. It is worth mentioning that the structure of such polyurethane-

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polyurea NP is finely tuned thanks to the specific components and assembling procedure employed. The high encapsulation efficiency (99%) of Plitidepsin in PUUa NP was attributed to

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further in vivo efficacy assays other hydrophobic drugs

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the high affinity of the drug for the oily core and ensured reaching the critical drug doses for . Indeed, high encapsulations have been obtained also for

. Interestingly, Plitidpesin encapsulation was highly stable, and

drug release only occurred in environments with high GSH content, such the cell cytosol. Moreover, the close to neutral surface charge of the nanoparticles, ensured a lower

release in bloodstream

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opsonization and accumulation in RES tissues

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. This specific design precluded the drug

and explained the increase in the MTD values obtained in vivo. It is

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worth emphasizing the fact that although the same drug dose was administered to mice in pharmacokinetic assays, the quantity of the drug in circulation was three orders of magnitude

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higher in the case of mice administered with the PUUa NP. Even with this increase in the circulating drug content, Plitidepsin loaded NP were less toxic in vivo than the free drug. The

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absence of toxicity of PUUa vehicle also helped. No toxicity was observed in vivo upon administration of the unloaded nanoparticles up to 1 g/kg of solid weight in mice. In vitro, we found that IC50 values of PUUa NP-RGD (without Plitidepsin loading) were around 10 µM, three orders of magnitude above those for Plitidepsin-loaded PUUa NP, indicating that these nanoparticles offer a good therapeutic window for treating oncologic diseases. Regarding size, PUUa NP had a polymeric dense morphology with monodisperse sizes around 90-110 nm (Figure 2, Table 1), which are between the optimal size range to achieve tumor accumulation in vivo by EPR and active targeting phenomena

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. Slightly lower sizes were

obtained for RGD-decorated NP, probably because the presence of the peptide adds extra hydrophilicity to the emulsification process. Neutral or slightly positive surface charges, as the one described for PUUa NP, have been reported as the most adequate for cell internalization

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.

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ACCEPTED MANUSCRIPT Indeed, our in vitro results demonstrate that PUUa NP enter the cells allowing the Plitidepsin release, and that this process was more effective for RGD-containing nanoparticles, at least after short incubation times. Recently, Goñi-de-Cerio et al.

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described the use of Plitidepsin

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loaded polymersomes targeted to the epidermal growth factor receptor (EGFR). They showed

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that the use of EGFR targeting increases the efficacy of the polymersomes by inducing a greater extent of apoptosis after 24 h incubation. However, as in our case, no clear advantages

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of the targeted and Plitidepsin loaded polymersome could have been observed in long-term cell proliferation MTT assays. Remarkably, we here found that the response of cells to PUUa NP

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could depend heavily on the cell type chosen. The MDA-MB-231 breast cancer cell line showed improved efficacy when treated with PUUa NP than with the free drug. The different responses could be related with the intrinsic endocytic capacity of each cell type and/or their relative levels of GSH. Indeed, MDA-MB-231 cell line have shown to have higher GSH levels than the other cell lines tested in this work, and this could well explain the higher effect of Plitidepsin loaded

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PUUa NPs, compared to the activity of the free Plitidepsin in this specific cell line. Nevertheless, the fine control of drug release in the presence of different levels of GSH should require further

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investigation.

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Interestingly, in the case of targeted nanoparticles, the linkage of the RGD through the amine residue leaves two charges per molecule, one positive and one negative, available in the

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surface of the nanoparticle, without altering the overall neutral surface charge. This zwitteronic characteristic of the RGD-decorated nanoparticles, could explain the differences in the biodistribution of targeted and non-targeted systems. Zwitteronic coatings reduce opsonization and the adsorption of a protein corona, because they prevent electrostatic interactions between circulating proteins and charged polymer surface and cancel the subsequent release of counterions and water molecules from nanoparticle surface

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. As a consequence, nanoparticles

with zwitterionic surfaces are known to escape the phagocytosis by RES. Kuppfer cells in the hepatic sinusoids of the liver and marginal zone and red pulpe macrophages in the spleen are the principal contributors to RES, and this likely explains the significantly lower accumulation of our targeted PUUa NP in these two organs. Moreover, this fact has been already demonstrated 6

for smaller size nanoparticles such as quantum dots . Sun et al., shown that the extent of uptake by the liver and spleen was about 3–6-fold lower for the zwitterionic quantum dots

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;

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ACCEPTED MANUSCRIPT and furthermore, Choi et al. also shown that the RGD functionalization can be achieved in nanoparticles

while maintaining their

zwitterionic characteristics

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. These optimized

biodistribution of the targeted nanoparticles, could also help to widen the therapeutic window of

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nanoparticles by reducing potential toxicities due to a lower degree of off-target binding. So far,

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previously described Plitidepsin-carrying nanosystems have shown little or none clear advantages over the standard formulation of Plitidepsin in Cremophor®:Ethanol:Water (CWE) in

PBLG or PTMC-b-PGA)

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in vivo efficacy assays. Polymer nanoparticles made of amphiphilic block copolymers (PEG-bor highly PEGylated poliglutamic acid (PGA) nanocapsules

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with

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Plitidepsin were not able to improve the tumor growth inhibition induced by the standard formulation of Plitidepsin. In this scenario, the use of targeted nanosystems that on the one hand facilitate the cell internalization nanoparticle is particularly relevant.

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and on the other improve the biodistribution of the

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It is worth mentioning that although polyurethanes and particularly polyureas are highly resistant to hydrolysis in aqueous media

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, PUUa NP were specifically designed to be lyophilizable

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and redispersable without the addition of any cryoprotectant or external surfactant and avoiding ultrasonication steps. To the best of our knowledge, this is the first time that PUUa crosslinked

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NP have been lyophilized and redispersed without any external surfactants or cryoprotectants. Thus, this finding is expected to, on the one hand, facilitate the handling and storage of PUUa

in vivo.

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NP, and on the other hand, avoid the use of cryoprotective agents that could cause side-effects

In summary, we herein report the use of PUUa NP for improving the toxicity, pharmacokinetic and biodistribution profile of a highly insoluble drug. The in vitro efficacy of Plitidepsin in different cancer cell lines was maintained when drug was non-covalently encapsulated in polyurethane-based nanosystems. In vivo, toxicity and pharmacokinetic profiles of Plitidepsin in PUUa was greatly improved when compared to the standard formulation of Plitidepsin. Moreover, functionalization of PUUa NP with cRGDfk moieties ameliorated the biodistribution of the nanosystem and the drug by avoiding their accumulation in tissues with high presence of macrophages such liver and spleen tissue. Overall, this study highlights the promising potential of RGD-targeted and non-targeted PUUa NP as delivery systems for anticancer drugs.

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Pegoretti A, Fambri L, Penati A, Kolarik J. Hydrolytic Resistance of Model Poly(ether urethane ureas) and Poly(ester urethane ureas)). J Appl Polym Sci. 1995;70:577-586. doi:10.1002/(SICI)1097-4628(19981017)70:3<577::AID-APP20>3.0.CO;2-X.

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Kozakiewicz J, Rokicki G, Przybylski J, Sylwestrzak K, Parzuchowski PG, Tomczyk KM. Studies of the hydrolytic stability of poly(urethane-urea) elastomers synthesized from oligocarbonate diols. Polym Degrad Stab. 2010;95:2413-2420. doi:10.1016/j.polymdegradstab.2010.08.017.

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ACCEPTED MANUSCRIPT FIGURE CAPTIONS Figure 1. PUUa NP synthetic process. Amphiphilic and hydrophobic prepolymers, lipophilic cargoes (drugs or lipophilic fluorophores) and cRGDfk targeting peptide are self-stratified in

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water by hydrophobic effects. After fixing the shell by interfacial crosslinking, the hydrophilic RGD targeting peptide is located at the surface of the PUUa NP allowing specific cell binding.

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Figure. 2. TEM micrographs of PUUa NP. A) PUUa NP-DiR; B) PUUa NP-DiR-PLI; C) PUUa NP-DiR-RGD; and D) PUUa NP-DiR-PLli-RGD. All nanoparticles showed a round spherical

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shape with very homogeneous sizes around 100 nm.

Figure 3. Degradation of PUUa NP. TEM micrograph proving the nanoparticle degradation process over time in the presence (A, B, C) or absence (D,E,F) of disulphide bonds in the polymeric shell. While disulfide-bearing NPs change their morphology over time in the presence

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of GSH, their disulfide-free counterparts remain stable under the same conditions. Figure 4. Cumulative Plitidepsin drug release from PUUa NP. Plitidepsin containing PUUa

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NP were incubated in degradation media (10 mM GSH, squares) or control media (no GSH, circles) and Plitidepsin drug concentrations were determined by HPLC. Mean ± SD are

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indicated with an n=2 in all cases.

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Figure. 5 In vitro efficacy of Plitidepsin-loaded PUUa NP in U87-MG and MDA-MB-231 cancer cell lines. MTT assays were performed after a long and continuous incubation during 72 h with PUUa (A and C) or for short-term 2 h incubation (B and D). To ensure the non-toxicity of the delivery system, non-loaded PUUa NP or non-conjugated cRGDfK PUUa NP were also included in the long-term MTT assays. Asterisks in B and D indicate significant or very significant differences between non-targeted and targeted nanoparticles after 2 h incubation (** for p<0.01 and *** for p<0.001). Data correspond to the mean of 3 independent experiments. Figure. 6. Pharmacokinetic profile of Plitidepsin in PUUa NP. Plasma concentration of Plitidepsin was determined after i.v. administration of 2 mg/kg Plitidepsin PUUa NP to CD-1 mice. Each point represents the mean and SD of n = 3.

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ACCEPTED MANUSCRIPT Figure 7. Liver accumulation of DiR-labelled PUUa NP in mice athymic mice. A) Noninvasive in vivo FLI images of integrin-targeted (PUUa NP-DiR-RGD) and non-targeted (PUUa NP-DiR-RGD) tissue biodistribution over time and B) semiquantitative estimation of

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fluorescence DiR intensity ratio in the liver area in vivo in respect to 1 h post administration. C)

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Ex vivo FLI liver images and D) quantification of liver DiR fluorescence per weight of tissue at 24 and 72 h end time points. Pseudocolor scale bars were consistent for all images in order to

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show relative changes with time.

Figure 8. Tissue biodistribution of integrin targeted and non-targeted PUUa NP loaded

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with Plitidepsin in athymic mice. Ex vivo plasma and tissue intensity per tissue weight at 72 h post administration of integrin targeted PUUa NP-DiR-PLI-RGD and non-targeted PUUa NP DiR-PLI at 1.7 mg PLI/kg and 0.4 mg DiR/kg by i.v. administration route is shown. Mean percentage values of each corresponding formulation tissue accumulation per tissue weight in

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respect to the total maximal tissue fluorescence are displayed in the graph. Data in the inserted table correspond to the mean ± standard deviation (SD) of Plitidepsin content measured by

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HPLC.

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Graphical Abstract

Polyurethane-polyurea nanoparticles (PUUa NP), with redox-responsive shell and RGD peptide

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functionalization, were synthetized incorporating a very lipophilic chemotherapeutic drug named Plitidepsin. This system offers several clear advantages: i) good efficacy and absence of vehicle

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toxicity, ii) fine control of the drug stability in plasma and a controlled release inside cancer cells, iii) RGD-functionalized PUUa NP showed an excellent in vivo biodistribution, avoiding their accumulation in tissues with high presence of macrophages, and iv) PUUa NP, consequently, improved very significantly the pharmacokinetic profile of Plitidepsin, offering a very promising platform for the delivery of specially difficult and lipophilic drugs.

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