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A new folate-grafted chitosan derivative to improve the delivery of paclitaxelloaded solid lipid nanoparticles for lung tumour therapy by inhalation Remi Rosiere, Matthias Van Woensel, Michel Gelbcke, Véronique Mathieu, Julien Hecq, Thomas Mathivet, Marjorie Vermeersch, Pierre G Van Antwerpen, Karim Amighi, and Nathalie Wauthoz Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00846 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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Molecular Pharmaceutics

A new folate-grafted chitosan derivative to improve the delivery of paclitaxel-loaded solid lipid nanoparticles for lung tumour therapy by inhalation Rémi Rosière1*, Matthias Van Woensel1,2, Michel Gelbcke3, Véronique Mathieu4, Julien Hecq1, Thomas Mathivet5, Marjorie Vermeersch6, Pierre Van Antwerpen7, Karim Amighi1 and Nathalie Wauthoz1 1

Laboratoire de Pharmacie Galénique et de Biopharmacie, Faculté de Pharmacie, Université

libre de Bruxelles (ULB), Brussels, Belgium 2

Research Group Experimental Neurosurgery and Neuroanatomy, Laboratory of Pediatric

Immunology, KULeuven, Leuven, Belgium 3

Laboratoire de Chimie Pharmaceutique Organique, Faculté de Pharmacie, ULB, Brussels,

Belgium 4

Laboratoire de Cancérologie et Toxicologie Expérimentale, Faculté de Pharmacie, ULB,

Brussels, Belgium 5

Institut National de la Santé et de la Recherche Médicale (INSERM), Unit 970, Paris Cardiovascular 6

Research Center, Paris, France

Center for Microscopy and Molecular Imaging (CMMI),

Gosselies, Belgium 7

Plateforme analytique de la Faculté de Pharmacie, Faculté de Pharmacie, ULB, Brussels,

Belgium *

Correspondence to: Rémi Rosière, Laboratoire de Pharmacie Galénique et de Biopharmacie,

Faculté de Pharmacie, Université libre de Bruxelles (ULB), Campus Plaine, CP207, Boulevard du Triomphe, B-1050 Brussels, Belgium. Tel +32 2 650 52 54. Fax +32 2 650 52 69; [email protected]

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ABSTRACT Inhaled chemotherapy for the treatment of lung tumours requires that drug delivery systems improve selectivity for cancer cells and tumour penetration and allow sufficient lung residence. To this end, we developed solid lipid nanoparticles (SLN) with modified surface properties. We successfully synthesized a new folate-grafted copolymer of polyethylene glycol (PEG) and chitosan, F-PEG-HTCC, with a PEG-graft ratio of 7% and a molecular weight range of 211-250 kDa. F-PEG-HTCC-coated, paclitaxel-loaded SLN were prepared with an encapsulation efficiency, mean diameter and zeta potential of about 100%, 250 nm and +32 mV, respectively. The coated SLN entered folate receptor (FR)-expressing HeLa and M109-HiFR cells in vitro, and M109 tumours in vivo after pulmonary delivery. The coated SLN significantly decreased the in vitro half maximum inhibitory concentrations of paclitaxel in M109-HiFR cells (60 vs 340 nM, respectively). We demonstrated that FR was involved in these improvements, especially in M109-HiFR cells. After pulmonary delivery in vivo, the coated SLN had a favourable pharmacokinetic profile, with pulmonary exposure to paclitaxel prolonged to up to 6 h and limited systemic distribution. Our preclinical findings therefore demonstrated the positive impact of the coated SLN on the delivery of paclitaxel by inhalation. KEYWORDS Active targeting, nanomedicine, lung carcinoma, controlled-release, pulmonary delivery ABBREVIATIONS 1

H-NMR, proton nuclear magnetic resonance spectroscopy; 25-NBD-cholesterol, 25-[N-[(7-

nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol;

DCC,

N,N’-

dicylclohexylcarbodiimide; DL, drug loading; DLS, dynamic light scattering; DMAP, 4(dimethylamino)pyridine;

DSC,

differential

scanning

calorimetry;

EDC,

N-(32

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dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride; EE, encapsulation efficiency; ESI-Q-TOF, electrospray ion source-quadrupole time-of-flight; FBS, foetal bovine serum; FR, folate receptor; HTCC, N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride; IC50,

half

maximum

inhibitory

concentration;

MTT,

3-[4,5-dimethylthiazol-2-yl]

diphenyltetrazolium bromide; Mw, molecular weight; MWCO, molecular weight cut-off; NH2-PEG-COOH, O-[2-aminoethyl]-O’-[3-carboxypropyl]polyethylene glycol 3000; NHS, N-hydroxysuccinimide; NSCLC, non-small cell lung carcinoma; PB5, phosphate buffer at pH 5; PBS, phosphate buffer saline; PdI, polydispersity index; PEG, poly(ethylene) glycol; PEGGR, poly(ethylene) glycol-graft ratio; PTX, paclitaxel; SLN, solid lipid nanoparticle; TEM, transmission electron microscopy; TGA, thermogravimetric analysis; TPGS, D-α-Tocopherol poly(ethylene glycol) 1000 succinate; Z-average, Z-average mean diameter



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1. INTRODUCTION Tumours localized in the respiratory tract remain an important public health issue. Primary lung tumours, classified as small cell carcinoma and non-small cell carcinoma (NSCLC), constitute the deadliest cancer, with 1.6 million deaths worldwide in 2012.1 Moreover, the lung is one of the most common sites of metastasis of tumours.2 A poor prognosis is associated with lung tumours, with a global 5-year survival rate of about 18% for primary tumours3 and from about 5 to 80% for pulmonary metastases, depending on the primary cancer.2 Therefore, research into the development of new effective treatment approaches is very active. These approaches include targeted therapies and immunotherapies able to treat well-defined subpopulations of patients, principally those with adenocarcinoma, which represents the main histology in NSCLC. These include, for example, 10-12% and 5% of patients with adenocarcinoma which express epidermal growth factor receptor mutations and express anaplastic lymphoma kinase rearrangement, respectively,4 and 23-28% of patients with advanced NSCLC which have a high level of programmed death ligand 1 expression.5 However, for most of patients with lung tumours, chemotherapy remains the backbone of the treatment modalities. This includes the use of platinum doublets composed of a platinum salt and another cytotoxic agent such as pemetrexed, gemcitabine, or a taxane derivative (e.g. paclitaxel (PTX)). To reduce the severe adverse effect observed with these agents, nanomedicine-based drug delivery systems have been described. An example of this system is nabTM-PTX (Abraxane®), which consists of PTX albumin-bound nanoparticles. Nab-PTX circumvents the issues related to the use of co-solvents with Taxol®, the conventional commercial form of the very poorly soluble compound PTX.6 It does so by exploiting the natural ability of PTX to bind albumin reversibly. This PTX-albumin binding induces a modification of the pharmacokinetic profile of PTX, with preferential accumulation of the nanoparticles in the tumour environment.7 Nab-PTX has reduced the toxicities of Taxol, with 4 ACS Paragon Plus Environment

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decreased neutropenia and less neuropathy observed.6,8 Consequently, higher PTX-related doses can be administered safely with nab-PTX compared to Taxol, resulting in a higher response rate in patients with advanced NSCLC.6,8,9 Another targeting approach is related to the use of the pulmonary route, which gives direct access to lung tumours. This concept now represents a realistic treatment alternative for certain populations of patients in terms of feasibility and safety.10–12 One of the main advantages of the pulmonary route over systemic routes is its better pharmacokinetic profile. Inhalation allows the administration of high drug doses directly to the tumour-bearing lung and the reduction of systemic distribution and toxicities, significantly enhancing the therapeutic ratio.13 However, despite clear advantages observed in Phase I14,15 and Phase Ib/IIa16–18 clinical trials, this approach has limited ability to go further, mainly because of technological challenges. The current inhalation devices and formulations used in clinical trials need to be adapted to deliver effective anticancer therapies to tumour-bearing lungs. Conventional immediaterelease drug formulations for inhalation (e.g. solutions for nebulization and soluble dry powders) can lead to too short residence in the lungs and to poor local tolerance. These are results of high peak concentrations of the anticancer drug in lung fluids after lung deposition.19–22 Therefore, one of the main challenges in the field is to develop inhalable formulations that progressively release the drug in the respiratory tract.23 In addition to flattening the local concentration peaks, these sustained-release formulations should maintain therapeutically-effective drug concentrations for a sufficient period of time throughout the lung tumour site.24,25 Consequently, an ideal formulation for inhalation should (i) release the anticancer drug progressively and (ii) avoid the non-absorptive clearance mechanisms in the respiration tract, e.g. the mucociliary escalator and alveolar and tumour-associated macrophages. 5 ACS Paragon Plus Environment


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Many formulation strategies have been proposed with this aim. Some of these are PEGylation of particle surfaces, for example on solid lipid microparticles23,26 or micelles,27 or coating particles with chitosan, for example as seen with chitosan-coated poly(lactic and glycolic acid) nanospheres.28 Chitosan is a biocompatible and biodegradable polymer composed of glucosamine and N-acetylglucosamine through β(1→4) glycosidic linkages. Chitosan is a polyamine compound soluble in acidic solutions. Below pH 6.5, the primary amines protonate, resulting in positively-charged soluble chitosan able to interact with the negativelycharged sialic acid of mucins. To avoid the pH dependence in solubilizing chitosan, derivatives have been described, e.g. trimethyl chitosan, carboxymethyl chitosan, and PEGylated chitosan. Chitosan derivatives are characterized by mucoadhesive properties that can prolong its residence in the lungs.28–30 Our research team have recently demonstrated mucoadhesive properties of N-[(2-hydroxy-3-trimethylammonium)propyl] chitosan chloride (HTCC)-coated liposomes in vitro.31 HTCC provided an increase in mucin adsorption of 22.9% at the liposome surface. In this context, we designed an HTCC derivative to modify the surface properties of solid lipid nanoparticles (SLN) containing PTX. This surface modification was hypothesized to prolong the retention of PTX within the lungs after inhalation. In addition, this chitosan derivative was thought to increase the selectivity of PTX-loaded SLN for lung cancer cells by actively targeting the folate receptors (FR) due to the presence of folate engraftments onto the polysaccharide backbone. FR, in particular its alpha form, are overexpressed onto the surface of lung tumour cells in many lung tumour types,32–37 e.g. about 72% of adenocarcinomas and 51% of squamous cell carcinomas,36 25% of SCLC, and 30% of lung metastases.37 FR-α is a membrane-associated form of FR that allows folate derivatives to bind, which permits their intracellular incorporation by endocytosis (i.e. receptor-mediated endocytosis).37–39 2. EXPERIMENTAL SECTION 6 ACS Paragon Plus Environment

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2.1.Materials 3-[4,5-dimethylthiazol-2-yl] diphenyltetrazolium bromide (MTT), 4-(dimethylamino)pyridine (DMAP),

dimethylsulfoxide

(DMSO)-d6, folic

acid,

N-(3-Dimethylaminopropyl)-N’-

ethylcarbodiimide hydrochloride (EDC), N,N’-dicylclohexylcarbodiimide (DCC), Nhydroxysuccinimide (NHS), sodium taurocholate, D-α-Tocopherol poly(ethylene glycol) 1000 succinate (TPGS), and triethylamine were purchased from Sigma-Aldrich (Diegem, Belgium). O-[2-aminoethyl]-O’-[3-carboxypropyl]polyethylene glycol 3000 (NH2-PEGCOOH) was purchased from Iris Biotech GmbH (Marktredwitz, Germany). HTCC was purchased from Kitozyme (Herstal, Belgium). Foetal bovine serum (FBS), gentamycin 50 mg/mL, penicillin-streptomycin 10 000 U/mL and Trypsin-EDTA 0.25% solutions, RPMI mediums, and phosphate buffer saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, USA). Cholesterol was purchased from Fagron (Waregem, Belgium). Cremophor® EL was purchased from BASF (Ludwigshafen, Germany). Glyceryl stearate (GeleolTM) was purchased from Gattefosse (Nanterre, France). The Matrigel® basement membrane matrix was purchased from Corning (Lasne, Belgium). PTX was purchased from Carbosynth (Berkshire, UK). All solvents (i.e. anhydrous DMSO, acetone, diethylether, acetonitrile) were purchased in analytical grade from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Purelab-Ultra device (Elga, Lane End, UK). 2.2.Synthesis of folate-poly(ethylene glycol)-N-[(2-hydroxy-3-trimethyl-ammonium) propyl] chitosan (F-PEG-HTCC) F-PEG-HTCC was synthesized using carbodiimide-mediated coupling chemistry40 adapted from different methods of F-PEG engraftment onto chitosan derivatives.41,42 2.2.1. Synthesis of folate-polyethylene glycol-COOH (F-PEG-COOH)



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The carboxylic groups of folic acid were first conjugated with the free primary amine of NH2PEG-COOH. Briefly, 1.1 g folic acid was dissolved in 15 mL anhydrous DMSO containing 700 µL triethylamine by sonication. Then 1 g DCC, 580 mg NHS, and 60 mg DMAP were added to this DMSO solution and the mixture was stirred at room temperature overnight in the dark. The precipitated by-product was eliminated by filtration and the filtrate was added dropwise to 350 mL of an anhydrous solution of acetone/diethyl ether (30:70 v/v) at 0°C. The yellow precipitate obtained was collected and washed with 3 × 20 mL of cold acetone/diethyl ether solution (30:70 v/v) to remove any trace of reagents and DMSO. This precipitate, corresponding to activated folic acid, was dissolved in 15 mL anhydrous DMSO containing 700 µL triethylamine, in the presence of 1.6 g NH2-PEG-COOH. This solution was stirred in the dark at room temperature for 24 h. The reaction mixture was first dialyzed against NaOH 0.1 M (molecular weight cut-off (MWCO) = 1 kDa, Spectra/Por, Spectrum Labs, Breda, the Netherlands) to eliminate DMSO, ultrafiltrated (MWCO = 1 kDa, Ultracell, Merck Millipore, Darmstadt, Germany) against NaOH 0.1 M and then against ultrapure water, and finally lyophilized by means of an Epsilon 1-6 freeze-dryer (Martin Christ GmbH, Osterode, Germany). The F-PEG-COOH molecular structure was confirmed by proton nuclear magnetic resonance spectroscopy (1H NMR) using a Bruker Avance 300 spectrometer (Bruker, Wissembourg, France) in DMSO-d6 at 25°C. The folate contents were determined in triplicate by quantitative UV spectrophotometry of the conjugate in methanol using the folic acid extinction coefficient ε value of 28 400 M-1 cm-1 at λmax of 285 nm using an Agilent 8453 UV/Visible Spectrophotometer (Agilent, Santa Clara, California, USA). 1H NMR (D2O) δ(ppm): 1.8 (2H, PEG, CH2CH2CH2COOH), 2.2 (2H, PEG, CH2COOH), 3.7 (288H, PEG, CH2O), 4.4 (1H, folate, glutamate), 4.6 (2H, folate, CH2NH), 6.9 (2H, folate, benzene), 7.7 (2H, folate, benzene), 8.7 (1H, folate, pteridin) (see also supplementary information, Fig. S1).


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The folate content value was 0.26 ± 0.01 mmol/g (98 ± 5% of the theoretical value, 0.27 mmol/g). 2.2.2. Grafting of F-PEG-COOH onto HTCC The free carboxylic group of F-PEG-COOH was afterwards coupled with the free primary amines of HTCC. Briefly, 420 mg HTCC (Mw = 92 kDa) was dissolved in 35 mL ultrapure water under magnetic stirring. Then 1.15 g F-PEG-COOH, 100 mg EDC and 75 mg NHS were added to this aqueous solution and the mixture was stirred at room temperature for 48 h. The reaction mixture was ultrafiltrated (MWCO = 30 kDa) against HCl 0.001 M and then lyophilized. The F-PEG-HTCC molecular structure was confirmed by 1H NMR. 1H-NMR of F-PEG-HTCC (D2O, 300 MHz, δ ppm): 2.1 (s, 3H, HTCC, -CO-CH3), 2.8 (s, 2H, HTCC, NH-CH2-CHOH-), 3.2 (s, 9H, HTCC, -N+(CH3)3), 3.3-4.2 (6H, glucosamine, C2-6HO), 3.7 (PEG, s, 288H, -CH2-O-), 4.3 (s, 1H, HTCC, -CH2-CHOH-CH2-), 6.9 (s, 2H, folate, benzene), 7.7 (s, 2H, folate, benzene), 8.8 (s, 1H, folate, pteridin). 2.3.Characterization of F-PEG-HTCC 2.3.1. Determination of the poly(ethylene glycol)-graft ratio (PEG-GR) The PEG-GR, defined as the number of PEG chains as a percentage of the total nitrogen number per F-PEG-HTCC molecule, was determined by 1H NMR using the following equation: − % = × × 9 ⁄ × + CH3 3 (1) where [PEG] is the integral of the proton peak of PEG at 3.7 ppm (CH2O); n H+ PEG is the number of protons per PEG chain according to the molecular weight (Mw) of PEG given by



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the supplier (Mw = 3317, n H+ PEG = 288); DQ is the degree of quaternization of HTCC (%); and [N+(CH3)3] is the integral of the proton peak at 3.2 ppm (N+(CH3)3). 2.3.2. Determination of the molecular weight range The Mw range of F-PEG-HTCC was determined by direct infusion of a solution in a 6520 series electrospray ion source (ESI)-quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). The acquisition mode was positive and the Mw range was obtained after deconvolution of the mass spectra. 2.3.3. Thermal properties – thermogravimetric analysis and differential scanning calorimetry The degradation temperature of F-PEG-HTCC was estimated by thermogravimetric analysis (TGA) using a Q500 apparatus (TA Instruments, Zellik, Belgium) and Universal Analysis 2000 version 4.4A software (TA Instruments). A run on about 5 mg copolymer was set on a platinum basket from 25°C to 300°C at a heating rate of 10°C/min. The thermal properties of F-PEG-HTCC were determined by differential scanning calorimetry (DSC) using a Q2000 differential scanning calorimeter coupled to a refrigerated cooling system (TA Instruments). Results were analysed using Universal Analysis 2000 version 4.4A software (TA Instruments). About 10 mg copolymer was placed in a Tzero hermetic aluminium pan. The heat run was set from 0°C to 190°C at 20°C/min using nitrogen as the blanket gas. 2.4. Preparation and characterization of PTX-loaded SLN coated with F-PEGHTCC PTX-loaded SLN coated with F-PEG-HTCC were prepared by a nanoprecipitation method at room temperature. First, PTX-loaded SLN were prepared. For this, a lipid solution containing 10 ACS Paragon Plus Environment

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10 mg glyceryl-stearate, 40 mg cholesterol, and 3 mg PTX in 2 mL acetone was prepared using an ultrasonic bath. The solution was poured into 20 mL ultrapure water containing 3 mg TPGS and 2 mg sodium taurocholate under magnetic stirring (1300 rpm). Precipitation immediately occurred. The SLN were then coated by adding 5 mL solution containing 7.5 mg F-PEG-HTCC in ultrapure water to the SLN dispersion. Acetone was finally evaporated at room pressure and temperature overnight. The SLN were analysed before and after coating at 25°C for particle size distribution (PSD), Z-average mean diameter (Z-average), and polydispersity index (PdI) by dynamic light scattering (DLS) and for zeta potential by laser Doppler electrophoresis using a Malvern Zetasizer nano ZS (Malvern Instruments SA, Worcestershire, UK). Each sample was analysed in triplicate and expressed as the mean ± SD. The morphology of PTX-loaded SLN (before and after F-PEG-HTCC coating) was evaluated by transmission electron microscopy (TEM) using the protocol described in our previous work.43 Briefly, 40 µL of nanosuspension was set onto a carbon-coated EM grid and stained with uranyl acetate (2%). After further blotting and drying, samples were directly observed on a Tecnai 10 TEM (FEI, Mérignac, France). Images were captured with a Veleta camera (Olympus Soft Imaging Solutions, Münster, Germany) and processed with iTEM (Olympus Soft Imaging Solutions) and Adobe Photoshop software. Before the F-PEG-HTCC coating, the PTX-loaded SLN dispersion was ultrafiltrated (MWCO = 10 kDa, Amicon©, Millipore) and the filtrates were collected. PTX encapsulation efficiency (EE, mass proportion relative to total PTX) and PTX drug loading (DL, mass proportion relative to excipients) were determined in triplicate. The quantification of PTX was determined by HPLC-UV analysis, as described in Section 2.8.2. Results are expressed as the mean ± SD (n=3).



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2.5.In vitro release profile of PTX The in vitro release profile of PTX from F-PEG-HTCC-coated SLN was determined in triplicate using the method described in our previous work.43 The dissolution medium was a phosphate buffer at pH 5 (PB5) (European Pharmacopoeia 7.0) to guarantee the stability of PTX.44 The PTX release from a Taxol-like formulation (i.e. 6 mg/mL PTX in a 50:50 v/v mix of Cremophor EL and absolute ethanol (≥99.8%)) in the same conditions was used as a control. Briefly, an amount of formulation corresponding to 20 µg PTX was added to PB5 to obtain a volume of 200 µL. The dispersion was put in a dialysis membrane (MWCO = 10 kDa) and placed in 200 mL PB5 maintained at 37 ± 1°C under magnetic stirring (200 rpm). The percentages of dissolved PTX that were released from the dialysis bag were determined using the HPLC method at different times up to 72 h. Results are expressed as the mean ± SD (n=3). 2.6.In vitro studies in FR-expressing cancer cell lines 2.6.1. Cell culture A human HeLa adenocarcinoma ovarian cell line (ATCC code CCL-2) and a murine M109HiFR lung carcinoma cell subline (kindly provided by Dr Hilary Shmeeda from Shaare Zedek Medical Center, Israel) were chosen for this study due to their high FR-α expression.45,46 FR-α protein expression was confirmed in the two cell lines by western blotting (see supplementary data). The two cell lines were cultured in folate-free RPMI supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.02% gentamycin in an atmosphere containing 5% CO2 at 37°C. 2.6.2. In vitro cell binding and uptake Cell binding and uptake of F-PEG-HTCC-coated SLN were evaluated in HeLa and M109HiFR cells using fluorescent microscopy and flow cytometry for qualitative and quantitative approaches, respectively. Fluorescent SLN, i.e. SLN containing 25-[N-[(7-nitro-2-1,312 ACS Paragon Plus Environment

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benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (25-NBD-cholesterol, i.e. a fluorescent dye) and coated with either F-PEG-HTCC, Alexa Fluor® 405-grafted F-PEG-HTCC (fluo-FPEG-HTCC), or PEG-HTCC were prepared with composition, size, and charge properties similar to PTX-loaded SLN coated with F-PEG-HTCC (see supplementary data). For fluorescent microscopy, cells were allowed to attach overnight in 6-well plates containing glass coverslips (5 x 104 and 1 x 105 cells per well for HeLa and M109-HiFR, respectively). After one wash with cold PBS (at about 4°C), cells were incubated for 30 min, 3 h, or 6 h at 37°C with fluo-F-PEG-HTCC-coated SLN or free 25-NBD-cholesterol, or left untreated as control conditions, in fresh folate-free RPMI. After two washes with cold PBS, cells were kept on ice until observation under a Zeiss Axio fluorescent microscope (Zeiss, Oberkochen, Germany). Two coverslips per condition were analysed. For flow cytometry analysis, cells were allowed to grow in 25 cm2 flasks for 24 h prior to the assay (0.6 or 1.2 x 106 cells per flask for M109-HiFR or HeLa, respectively). After one wash with cold PBS (at about 4°C), cells were incubated for 30 min, 3 h or 6 h at 37°C with FPEG-HTCC-coated SLN or PEG-HTCC-coated SLN, or left untreated as control conditions, in fresh folate-free RPMI, or with F-PEG-HTCC-coated SLN in fresh medium supplemented with 1 mM folic acid. After the indicated incubation period, cells were washed once with cold PBS, detached by trypsin-EDTA treatment, centrifuged, and suspended in 1 mL cold PBS. Cell suspensions were kept on ice until analysis with a flow cytometer Quanta SC flow (Beckman Coulter Analis, Suarlée, Belgium). Results are expressed as mean green fluorescent signal (FL1 mean) per 10 000 cells, which is directly proportional to the number of SLN interacting with cells. Each condition was realized in triplicate and results are expressed as the mean ± SEM. 2.6.3. In vitro anti-proliferative properties



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The anti-proliferative properties of F-PEG-HTCC-coated SLN were determined by means of the colorimetric MTT assay in M109-HiFR and HeLa cells using an adaptation of the reported protocol.43 The main adaptation concerned the nanoparticles evaluated, i.e. PTX-loaded SLN (F-PEG-HTCC-coated or PEG-HTCC-coated SLN) or empty SLN. A Taxol-like formulation (as prepared in Section 2.5) was used as a positive control. All the formulations were incubated for 8 h and the cells were let to grow for 64 additional hours. The amount of formazan measured using a Biorad 680XR spectrometer (Biorad, Nazareth, Belgium) at 570 nm (with a reference of 630 nm) was directly proportionate to the number of living cells. The half maximum inhibitory concentration (IC50) values (relative to PTX) were defined as the PTX concentrations that inhibit 50% of cell growth compared to the untreated control. The experiments were performed three times (from independent formulations and cell batches) in six replicates. Results are expressed as the mean ± SEM. 2.7.Animals and husbandry conditions Female CD1 (Janvier Labs, Le Genest-Saint-Isle, France) and female BALB/c mice (Charles River, Arbresle, France) were kept under conventional housing conditions (22 ± 2°C, 55 ± 10% humidity, and 12-hour day/night cycle). All studies and manipulations were approved by the CEBEA ethical committee of the Faculty of Medicine, ULB (Commission d’Ethique du Bien-Etre Animal, Université libre de Bruxelles, Belgium) (Ethical protocols N°424N, 569N, and 585N). The laboratory federal agreement number is LA 1230568. 2.8.Pulmonary and systemic exposures to PTX 2.8.1. PTX delivery to mice Pulmonary and systemic exposures to PTX were evaluated in mice after the administration of PTX-loaded formulations. A dose related to 1 mg/kg PTX was administered to mice as (i) FPEG-HTCC-coated SLN delivered by the endotracheal route (50 µL), (ii) a Taxol-like 14 ACS Paragon Plus Environment

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formulation (as prepared in Section 2.5) delivered by the endotracheal route (50 µL), and (iii) a Taxol-like formulation delivered by the intravenous route (200 µL, tail vein). This dose was safe by the non-invasive endotracheal route with body weight losses that did not exceed 5% (see supplementary information, Fig. S4).The formulations delivered by the non-invasive endotracheal route were administered using an aerosolizing system (MicrosprayerTM model IA-1C®, Penn-Century, Philadelphia, USA) and using the protocol described by Wauthoz et al.47 The mice were euthanized by a lethal intraperitoneal injection of 12 mg sodium phenobarbital (Nembutal®, Ceva Santé Animale, Brussels, Belgium) immediately (0 h) and after 1 h and 6 h. Blood was collected from the orbital sinus in heparin-lithium tubes (Sarstedt AG & Co, Nümbrecht, Germany). Blood samples were centrifuged at 2000 g at room temperature and the supernatants (i.e. plasma) were preserved at -20°C until PTX extraction (see Section 2.8.2). The lungs were removed, immediately put in liquid nitrogen and preserved at -20°C until PTX extraction. 2.8.2. Determination of PTX content in lung tissues and plasma PTX was first extracted from lung tissues and plasma. Lung tissues were powdered in liquid nitrogen using a mortar and pestle, weighed, and vortexed in NaCl 0.9% (1:1 w/w) for 1 min. A volume of acetonitrile was added to plasma or tissue homogenates (2:1 v/v). The samples were vortexed for 5 min and then centrifuged at 15 000 g for 20 min at 4°C. The supernatants were collected and kept at -20°C prior to HPLC analysis. PTX in the supernatants was quantified using an adaptation of the HPLC method described in our previous work.43 Briefly, the HPLC system (HP 1200 series, Agilent Technologies) was equipped with a quaternary pump, an auto sampler, and a variable wavelength detector. The separations were performed on a reverse-phase Hypersil© Gold C-18 column (5 µm, 250 mm x 4.6 mm) (Thermo Fisher Scientific) at 30°C. The mobile phase consisted of ultrapure



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water/acetonitrile (47:53 v/v), which was delivered at a flow rate of 1.0 mL/min. The quantification was performed at 227 nm, the volume injected was 20 µL, and the analysis run time 10 min. The limits of detection and quantification were respectively 80 ng/mL and 250 ng/mL in plasma, and respectively 0.3 µg/g and 2 µg/g in lung tissues. Linearity was confirmed in the PTX concentration range of 250-5000 ng/mL and 2-120 µg/g in plasma and lung tissues, respectively. Recoveries within the calibration curve were 93 ± 13% and 105 ± 4% in plasma and lung tissues, respectively (mean ± SD, n=3). 2.9.SLN distribution into solid lung tumours The distribution of F-PEG-HTCC-coated SLN into M109 lung tumours was evaluated after pulmonary delivery using the protocol described previously.43 Briefly, on day thirteen post M109-HiFR cell-intrapulmonary implantation, 25-NBD cholesterol-loaded SLN coated with fluo-F-PEG-HTCC (see Section 2.6.2) were administered to mice through the endotracheal route. An amount of 50 µg far-red fluorescent Alexa Fluor® 647 isolectin GS-IB4 conjugate (Thermo Fischer Scientific) was then injected intravenously. After 2 h, the lungs were collected and tumour tissues were isolated. The tissues were incubated overnight in the dark in 4% formaldehyde in PBS (solution/tissue ratio of at least 10:1) and finally cut into ~1 mm slices using a scalpel. Three slices were cut throughout the tumours. The slices were mounted on SuperFrostTM slides (Thermo Fischer Scientific) using a fluorescent mounting medium (Dako, Heverlee, Belgium) and finally covered with a coverslip. The slides were observed under a Leica SP8 confocal microscope (Leica Microsystems, Diegem, Belgium). The images were processed using ImageJ software (National Institutes of Health, USA). 2.10.

Statistical analysis


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The data were analysed using GraphPad Prism software (version 5.01). All statistical tests used are indicated in Section 3. 3. RESULTS 3.1.Synthesis of the new targeted chitosan copolymer F-PEG-HTCC A new folate-grafted N,N,N-trimethyl chitosan derivative conjugate of three components, i.e. F-PEG-HTCC, was successfully synthesized. As presented in Fig. 1A, F-PEG-HTCC is composed of three main parts: (i) the HTCC chains, on which are grafted (ii) the PEG chains and (iii) the folate groups at the PEG chain extremities. F-PEG-HTCC was synthesized using carbodiimide-mediated coupling chemistry in two main steps. The first step consisted of the coupling of folate group and NH2-PEG-COOH through the formation of an amide linkage. The chemical structure and folate content of F-PEG-COOH were determined using 1H NMR and UV spectroscopy, respectively. The folate content was 0.26 ± 0.01 mmol/g (98 ± 5% of the theoretical value, 0.27 mmol/g) (mean ± SD). The second step consisted of grafting FPEG-COOH onto HTCC chains though the formation of amide linkages. The chemical structure of F-PEG-HTCC was confirmed using 1H NMR. This showed characteristic peaks at 3.2 ppm and 3.7 ppm, corresponding to the trimethylated ammonium group (i.e. in HTCC) and PEG, respectively (Fig. 1B). PEG-GR was determined from the 1H NMR spectra and was 7% (Fig. 1C). The Mw range was 211-250 kDa (Fig. 1C), confirming the successful grafting of PEG chains onto the HTCC backbone. Relative abound of m/z after deconvolution of the mass spectra of F-PEG-HTCC is presented in supplementary information (Table S1).This wide range could be explained by random grafting of PEG chains onto the HTCC backbone, leading to a mixture of grafted HTCC.



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Figure 1. Characterization of F-PEG-HTCC. (A) Chemical structure, (B) 1H NMR spectra in D2O, (C) physicochemical characteristics obtained by H1 NMR and ESI-Q TOF mass spectrometry, (D) thermogram obtained by TGA at 10°C/min, and (E) DSC curve at 20°C/min in a Tzero hermetic aluminium pan. TGA analysis of F-PEG-HTCC revealed two slopes of degradation (Fig. 1D). The first slope started at ~200°C and represented ~30% of mass loss. This first degradation should therefore correspond to the degradation of the HTCC backbone, considering its mass proportion in FPEG-HTCC (i.e. about 36-42%) and data from the literature.48 The second slope of degradation was observed at higher temperatures (from approximately 320°C) and was thus 18 ACS Paragon Plus Environment

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attributed to the PEG moiety.48 DSC analysis showed endothermic peaks at 53°C and 133°C (Fig. 1E), which were attributed to the melting points of PEG and HTCC moieties, respectively (provided by the suppliers). 3.2.Physicochemical characteristics of PTX-loaded SLN coated with F-PEG-HTCC Table 1 presents the physicochemical properties of PTX-loaded SLN, before and after coating with F-PEG-HTCC. Uncoated PTX-loaded SLN presented a monodispersed PSD (Fig. 2A) that was characterized by a Z-average of 165 ± 8 nm and a PdI of 0.18 ± 0.04. The zeta potential was -17 ± 1 mV. The EE was 99.0 ± 0.3% (w/w) and the DL was 5.1 ± 0.2% (w/w). This high drug encapsulation value was attributed to the lipophilic nature of PTX, which facilitated its encapsulation into a lipid matrix. Table 1. Z-average, polydispersity index (PdI), zeta potential, encapsulation efficiency (EE), and drug loading (DL) of the PXT-loaded SLN, uncoated and coated with F-PEG-HTCC. Data are expressed as the mean ± SD (n=3). ‘nd’ means not determined. Formulation

Z-average (nm)

PdI

Zeta potential (mV)

EE (% w/w)

DL (% w/w)

Uncoated SLN

165 ± 8

0.18 ± 0.04

-17 ± 1

99.0 ± 0.3 5.1 ± 0.2

F-PEG-HTCC-coated SLN

249 ± 36

0.31 ± 0.02

+32 ± 1

99.0 ± 0.3 4.6 ± 0.1



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Figure 2. Characterization of the PTX-loaded SLN, uncoated and coated with F-PEG-HTCC. (A) PSD curves of uncoated (red curve) and F-PEG-HTCC-coated (blue curve) PTX-loaded SLN, (B) TEM images (bar scales of 200 nm, the white arrows indicate SLN cores), and (C) in vitro release profiles of PTX in PB5 at 37°C. Formulations containing 20 µg PTX were put in a dialysis membrane (MWCO = 10 kDa) and placed in 200 mL of dissolution medium under stirring (200 rpm) (sink conditions). Data represent the mean ± SD (n=3) Efficient coating was confirmed by both an increased Z-average (i.e. from approximately 165 nm to 250 nm before and after coating, respectively) and positive zeta potential values (i.e. from approximately -17 mV to +32 mV before and after coating, respectively) (Table 1). The 20 ACS Paragon Plus Environment

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coating is related to the presence of opposite charges in water, with the negatively charged lipid particle surface and the positively charged F-PEG-HTCC. The coating affected the PdI, with higher values for the coated than for the uncoated SLN. The DL after coating was 4.6 ± 0.1% (w/w) (Table 1). TEM images (Fig. 2B) revealed a spherical morphology for PTX-loaded SLN. The nouniform distribution of uncoated SLN (Fig. 2B) was explained by the fact that some parameters of the nanoprecipitation method (e.g. the addition rate of acetone in water) were uncontrolled at lab-scale. After coating with F-PEG-HTCC, the SLN appeared fully surrounded in a swollen structure. This structure was composed of an F-PEG-HTCC copolymer and fully surrounded PTX-loaded SLN. However, these TEM results showed no SLN aggregation when dispersed in water. SLN were observed in dried samples obtained after evaporation of water in which they were dispersed. In this case, TEM analysis does therefore not confirm, in contrast with DLS, that the SLN were coated when dispersed in water. TEM analysis confirmed the size and spherical morphology preservation of PTXloaded SLN after F-PEG-HTCC coating. 3.3.In vitro release profile of PTX from F-PEG-HTCC-coated SLN Fig. 2D shows a cumulative PTX release from F-PEG-HTCC-coated SLN of about 50% within 3 days. After a first burst (13% of PTX released within 7 h), the PTX release rates remained constant, with approximately 15% of the PTX released each 24 h. Conversely, the PTX release from the Taxol-like formulation was much faster, with about 70% of the PTX released within 24 h, which is similar to that previously reported.49 It must be noted that, for the coated SLN, we were not able to study the burst effect in more detail. This was because, for the amount of PTX involved and the volume of the dissolution medium (required to maintain sink conditions), no quantification was possible with the HPLC method below 7 h.



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3.4.In vitro cell binding and uptake As shown in fluorescent images in Fig. 3A, the coated SLN entered, or at least bound, both the HeLa and the M109-HiFR cells, which are two FR-expressing cancer cell lines (see supplementary data). Green (corresponding to 25-NBD cholesterol) and blue (corresponding to fluo-F-PEG-HTCC) fluorescence seemed to increase with the time of incubation, especially regarding differences in results obtained between 30 min and 3 h incubation. It was observed that free 25-NBD cholesterol also stained the cells because of its very high affinity for the cells (supplementary data). Although auto-fluorescence was observed in untreated controls (especially in M109-HiFR, Fig. 3A), much higher blue fluorescence (i.e. corresponding to the copolymer coating) was observed for cells incubated with the coated SLN.


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Figure 3. In vitro cell binding and uptake. (A) Fluorescent images of HeLa and M109-HiFR cells following the incubation of fluo-F-PEG-HTCC-coated SLN loaded with 25-NBDcholesterol for 30 min, 3 h and 6 h (green = 25-NBD-cholesterol; blue = Alexa Fluor® 405grafted-F-PEG-HTCC, bar scales of 20 µm); (B) flow cytometry analysis. Quantification of green fluorescence (FL1 mean) of HeLa and M109-HiFR cells, untreated (control, white), and following the incubation of 25-NBD cholesterol-loaded SLN uncoated (beige) or coated with 23 ACS Paragon Plus Environment


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PEG-HTCC (blue) or F-PEG-HTCC (red) incubated in folate-free RPMI, and F-PEG-HTCCcoated SLN incubated in the presence of 1 mM folic acid (barred red) for 30 min, 3 h, and 6 h. The data are expressed as the mean ± SEM (n=3). In flow cytometry analysis, green fluorescence corresponding to 25-NBD cholesterol was quantified. In contrast to very low auto-fluorescence in control cells (FL1 mean of about 6), green fluorescence was detected in treated cells regardless of the time or conditions of incubation (Fig. 3B). These data therefore confirmed cell uptake, or at least cell binding, of the SLN, as observed in fluorescence microscopy. This was already the case after 30 min incubation (for instance, the FL1 mean values observed after 30 min incubation of HeLa with F-PEG-HTCC-coated SLN and the control were 11 and 5, respectively). Moreover, regardless of the evaluated condition and the cell line, the longer the time of incubation, the higher the binding/incorporation was (for instance, FL1 mean values for F-PEG-HTCC-coated SLN in contact with M109-HiFR were 19, 28, and 39 after 30 min, 3 h, and 6 h incubation, respectively) (Fig. 3B). Another observation was that, following the same time of incubation in the same cell line, most of the evaluated conditions led to similar green fluorescence and therefore similar binding/uptake (Fig. 3B) (for instance, FL1 means of ~18 after 3-h incubation in HeLa). The influence of FR-involved mechanisms therefore seemed to be low. An explanation would be that, in addition to FR-related mechanisms, the SLN (i.e. both F-PEG-HTCC-coated and PEG-HTCC-coated SLN) bind the cells via additional mechanisms50 PEG-HTCC-coated SLN indeed present excellent properties for entering living cells regarding their size (i.e. significant particle populations with a diameter less than 150 nm, see supplementary data), surface (i.e. PEGylated nanoparticles), and charges (i.e. positively charged HTCC). However, the binding/uptake of F-PEG-HTCC-coated SLN into M109-HiFR slightly decreased in the


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presence of free folic acid in the incubation medium (Fig. 3B), suggesting the involvement of a folate-related pathway, possibly FR-mediated endocytosis. An interesting observation was that, depending on the cell line, different levels of cell binding/uptake were obtained. SLN led to higher levels of binding/uptake into M109-HiFR than into HeLa (FL1 means of 40 and 20, respectively, after 6 h incubation with F-PEGHTCC-coated SLN). The mechanisms of entry involved (i.e. both folate and non-folaterelated) were therefore highly cell-line dependent, as commonly assumed.50,51 3.5.In vitro anti-proliferative properties of F-PEG-HTCC-coated SLN Regarding the results obtained (Fig. 4), the first observation was that HeLa cells were significantly more sensitive to PTX-based formulations than M109-HiFR (p < 0.01, two-way ANOVA). For instance, the positive control Taxol was approximately ten times more active in HeLa than in M109-HiFR (IC50 of 35 and 340 nM, respectively, Fig. 4). These results were consistent with those from literature.52,53

Figure 4. Half maximum inhibitory concentration (IC50), determined by means of the colorimetric MTT assay. HeLa and M109HiFR cells were incubated with Taxol (positive control in light grey), PTX-loaded SLN uncoated (beige) or coated with PEG-HTCC (blue) or F-PEG-HTCC (red) were incubated in folate-free RPMI or in the presence of 1 mM folic acid 25 ACS Paragon Plus Environment


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(barred red) for 8 h. The data are presented as mean IC50 values ± SEM from three independent tests realized in six replicates. (*) p < 0.05; (**) p < 0.01; (***) p < 0.001; ‘n.s.’ means non-significant (two-way ANOVA with Bonferroni post-tests). The anti-proliferative properties of the SLN was highly dependent on the cell line (p < 0.01, two-way ANOVA). Whereas F-PEG-HTCC-coated SLN led to an IC50 similar to that of Taxol in HeLa (about 40 nM), in M109-HiFR, they increased very significantly the anti-proliferative properties of PTX (p < 0.001, two-way ANOVA with Bonferroni post-tests, Fig. 4). In M109HiFR, the IC50 was reduced almost 6-fold compared to Taxol (60 and 340 nM for F-PEGHTCC-coated SLN and Taxol, respectively, Fig. 4). F-PEG-HTCC-coated SLN were almost five times more active than PEG-HTCC-coated SLN in M109-HiFR (IC50 values of 60 and 295 nM, respectively) and almost three times more active in HeLa (45 and 126 nM, respectively) (Fig. 4). This difference was significant in M109-HiFR but not in HeLa (p < 0.01 and p > 0.05, respectively, two-way ANOVA with Bonferroni post-tests, Fig. 4). The IC50 values of F-PEG-HTCC-coated SLN were at least doubled in the presence of folic acid (170 and 130 nM in HeLa and M109-HiFR, respectively). However, these IC50 increases were not significant (p > 0.05, two-way ANOVA with Bonferroni post-tests, Fig. 4). No significant differences in the percentage of viable cells were observed with empty F-PEGHTCC-coated SLN for the concentrations evaluated in both cell lines, compared to untreated control cells (p > 0.05, one-way ANOVA, data not shown). 3.6.Pulmonary and systemic exposure to paclitaxel Pulmonary delivery of PTX (as in Taxol as well as in the coated SLN) led to higher pulmonary concentrations several minutes following the administration procedure, i.e. at time = 0 h (Fig. 5A). The pulmonary concentrations determined correspond to PTX, either as free 26 ACS Paragon Plus Environment

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dissolved PTX or as contained in the SLN. This difference was very significant for the coated SLN but not for Taxol (p < 0.01 and > 0.05, respectively, one-way ANOVA with Dunn’s post-tests). However, for longer time periods, the two routes of administration (i.e. inhalation and systemic routes) evaluated with Taxol led to similar pulmonary concentrations (5-10 µg/g and 1 µg/g after 1 and 6 h, respectively). In contrast, inhaled F-PEG-HTCC-coated SLN led to 7-fold (p > 0.05, one-way ANOVA with Dunn’s post-tests) and 32-fold (p < 0.05, one-way ANOVA with Dunn’s post-tests) increased concentrations compared to inhaled Taxol after 1 and 6 h, respectively.

Figure 5. Pulmonary (A) and systemic (B) exposure to PTX following the administration of intravenous Taxol (black), inhaled Taxol (grey), and inhaled F-PEG-HTCC-coated SLN (green). The data are presented as mean values ± SEM (n=5-6); (*), p < 0.05; (**), p < 0.01 (Kruskall-Wallis with Dunn’s post-tests); ‘

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