Accepted Manuscript Title: Hybrid nanosystems based on natural polymers as protein carriers for respiratory delivery: Stability and toxicological evaluation Author: Susana Rodrigues Clara Cordeiro Bego˜na Seijo Carmen Remu˜na´ n-L´opez Ana Grenha PII: DOI: Reference:

S0144-8617(15)00079-X http://dx.doi.org/doi:10.1016/j.carbpol.2015.01.048 CARP 9636

To appear in: Received date: Revised date: Accepted date:

18-8-2014 27-11-2014 21-1-2015

Please cite this article as: Rodrigues, S., Cordeiro, C., Seijo, B., Remu˜na´ n-L´opez, C., and Grenha, A.,Hybrid nanosystems based on natural polymers as protein carriers for respiratory delivery: Stability and toxicological evaluation, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.01.048 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights:  CS/CRG/TPP nanoparticles exhibit a biocompatible profile in respiratory epithelial cells  The nanoparticles are stable in presence of lysozyme

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 Glucose and sucrose are effective cryoprotectants of nanoparticles during freezedrying

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Hybrid nanosystems based on natural polymers as protein carriers for respiratory

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delivery: Stability and toxicological evaluation

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Susana Rodriguesa, Clara Cordeirob,c,d, Begoña Seijoe, Carmen Remuñán-Lópeze, Ana Grenhaa*

CBME – Centre for Molecular and Structural Biomedicine / IBB – Institute for

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a

Biotechnology and Bioengineering, University of Algarve, Faculty of Sciences and

Faculty of Sciences and Technology, University of Algarve, Campus de Gambelas, 8005-

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Technology, Campus de Gambelas, 8005-139 Faro, Portugal;

139 Faro, Portugal;

CEAUL- Center of Statistics and Applications, Faculty of Sciences, University of Lisbon,

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Campo Grande, 1749-016 Lisboa, Portugal;

NanoBioFar Group, Department of Pharmacy and Pharmaceutical Technology, Faculty of

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CESUAlg- Centre for research and development in Health, University of Algarve, Portugal;

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Pharmacy, University of Santiago de Compostela, Campus Vida, 15782 Santiago de

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Compostela, Spain

Author e-mails (per order): [email protected], [email protected], [email protected], [email protected], [email protected]

*Corresponding author:

E-mail address: [email protected] Postal address: Centre for Molecular and Structural Biomedicine (CBME), Faculty of Sciences and Technology, Building 8, Room 3.1, Campus de Gambelas, 8005-139 Faro, Portugal; Phone: +351 289800100 - Ext. 7441; Fax: +351 289 818419

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Abstract

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Chitosan/carrageenan/tripolyphosphate nanoparticles were previously presented as holding

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potential for an application in transmucosal delivery of macromolecules, with

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tripolyphosphate demonstrating to contribute for both size reduction and stabilisation of the

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nanoparticles. This work was aimed at evaluating the capacity of the nanoparticles as protein

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carriers for pulmonary and nasal transmucosal delivery, further assessing their

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biocompatibility pattern regarding that application. Nanoparticles demonstrated stability in

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presence of lysozyme, while freeze-drying was shown to preserve their characteristics when

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glucose or sucrose were used as cryoprotectants. Bovine serum albumin was associated to the

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nanoparticles, which were successfully microencapsulated by spray-drying to meet the

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aerodynamic requirements inherent to pulmonary delivery. Finally, a satisfactory

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biocompatibility profile was demonstrated upon exposure of two respiratory cell lines (Calu-

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3 and A549 cells) to the carriers. A negligible effect on cell viability along with no alterations

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on transepithelial electrical resistance and no induction of inflammatory response were

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observed.

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Keywords: biocompatibility; microencapsulation; nanoparticles; nasal delivery; protein

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delivery; pulmonary delivery

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1. Introduction

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Several physicochemical and biopharmaceutical issues make systemic administration of

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biomolecules a challenging task (Gupta & Sharma, 2009), compelling the need to develop

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adequate drug delivery systems that overcome inherent limitations. These carriers are mainly

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developed to avoid the need of parenteral administration, instead permitting effective

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transmucosal delivery. The pulmonary and nasal routes are increasingly addressed in this

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context, because of the large absorptive surface and the easy access, respectively, among

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other characteristics. Carriers are thus designed to exhibit characteristics that improve the

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epithelial contact, while protecting sensitive bioactive materials from in vivo degradation.

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Nanoparticles are first-line candidates for this end, successfully associating therapeutic

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macromolecules (Pinto Reis, Neufeld, Ribeiro & Veiga, 2006). A previous study

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demonstrated the ability of two natural polymers, chitosan (CS) and κ-carrageenan (CRG), to

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assemble into nanoparticles of 400-600 nm by simple polyelectrolyte complexation (Grenha

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et al., 2010). While CS is a very well-known material, the abilities of CRG are not

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sufficiently explored. Apart from the advantage of using natural materials, which facilitates

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biocompatibility (Malafaya, Silva & Reis, 2007), the polyelectrolyte complexation between

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CS and CRG uses very mild conditions, occurring in hydrophilic medium (Grenha, 2012).

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Nevertheless, the intimate contact between the carrier and the epithelial surface that highly

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favours mucosal delivery of drugs is reported to be maximal for particles of 50-500 nm

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(Desai, Labhasetwar, Amidon & Levy, 1996; Jani, Halbert, Langridge & Florence, 1990) and

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strong positive surface charge (Bogataj et al., 2003; Carvalho, Bruschi, Evangelista &

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Gremião, 2010). Therefore, more recently we associated tripolyphosphate (TPP) to the

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CS/CRG nanoparticle formulation as a cross-linking agent, providing a size decrease and

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obtaining more stable nanoparticles (Rodrigues, Rosa da Costa & Grenha, 2012).

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Notwithstanding the relevance that nanoparticles have gained in the last decade, the fact is

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that several limitations are still to overcome regarding their application. The inherent

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tendency for particle aggregation, chemical instability of polymers and early release of active

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substances are major problems to be addressed. In addition, as nanoparticles are usually

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formulated as suspensions, they are prone to microbial growth (Wu, Zhang & Watanabe,

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2011). Therefore, under all aspects, the physical and chemical stability of the systems is

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expected to be improved if water is removed (Abdelwahed, Degobert, Stainmesse & Fessi,

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2006). The shelf-life of nanoparticles is a relevant aspect of their potential application as drug

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delivery carriers, especially concerning the need to maintain unaltered physicochemical

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characteristics along time. Freeze-drying appears in this context as a very adequate choice,

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ensuring a soft drying procedure. The more aggressive conditions associated to freeze-drying

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can be softened by the use of specific protectants. These will provide the formation of a solid

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amorphous glass film around the nanoparticles and the immobilisation of the carriers,

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preventing aggregation and promoting stabilisation(Beirowski, Inghelbrecht, Arien &

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Gieseler, 2011, 2012).

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Safety issues are another permanent subject adjacent to nanoparticle applications. In fact,

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regarding the inhalation of nanoparticles, their fate after administration remains uncertain and

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there is the possibility of accumulation in the lung tissue upon repeated administrations

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(Rogueda & Traini, 2007). With regard to the evaluation of materials safety, there is a general

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recommendation considering that an initial in vitro evaluation should comprise different cell

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lines and times of exposure, while not only cell viability but also the inflammatory response

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should be determined (ISO, 2009; Rodrigues, Dionísio, Remuñán-López & Grenha, 2012).

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The Calu-3 and A549 respiratory epithelial cell lines have been frequently used in this

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context to evaluate the behaviour of systems aimed at respiratory drug delivery, either nasal

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or pulmonary (Foster, Avery, Yazdanian & Audus, 2000; Grenha et al., 2007; Nahar et al.,

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2013).

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In this work, we aimed at exploring the potential of CS/CRG/TPP nanoparticles for an

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application in nasal and pulmonary transmucosal delivery of proteins, placing a particular

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emphasis on their biocompatibility behaviour. Additionally, the stability of nanoparticles in

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presence of lysozyme (an enzyme present in the nasal and lung mucosa), as well as the ability

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of freeze-drying to provide a dry form of nanoparticle suspensions, were also assessed.

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Regarding pulmonary administration, as nanoparticles are known to lack the adequate

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aerodynamic diameter to permit deep lung delivery with success, which should vary between

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1 and 5 µm, the strategy of microencapsulating the nanocarriers was applied, following a

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previously described approach (Grenha, Seijo & Remuñán-López, 2005).

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2. Materials and Methods

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2.1. Materials

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Chitosan (low molecular weight, deacetylation degree = 75-85%), pentasodium

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tripolyphosphate, bovine serum albumin (BSA), phosphate buffer saline (PBS) tablets pH

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7.4, Dulbecco’s modified Eagle’s medium (DMEM), penicillin/streptomycin (10000

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units/mL, 10000 µg/mL), non-essential amino acids, L-glutamine 200 mM, trypsin–EDTA

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solution (2.5 g/L trypsin, 0.5 g/L EDTA), trypan blue solution (0.4%), thiazolyl blue

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tetrazolium bromide (MTT), sodium dodecyl sulphate (SDS), N,N-dimethylformamyde

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(DMF), glycerol, lipopolysacharide (LPS), dimethyl sulfoxide (DMSO), lysozyme and

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glacial acetic acid were supplied by Sigma Chemicals (Germany). k-carrageenan was

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obtained from FMC Biopolymer (Norway) and fetal bovine serum (FBS) was from Gibco

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(USA). Ultrapure water (Milli-Q Plus, Millipore Iberica, Spain) was used throughout.

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2.2 Cell lines

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The Calu-3 and A549 cell lines were obtained from the American Type Culture Collection

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(Rockville, USA) and used between passages 35–45 and 20-30, respectively. Cell cultures

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were grown in 75 cm2 flasks in a humidified 5% CO2/95% atmospheric air incubator at 37 ºC.

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For both cell lines, cell culture medium (CCM) was DMEM supplemented with 10% (v/v)

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FBS, 1% (v/v) non-essential amino acids solution, 1% (v/v) L-glutamine solution (200 mM)

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and 1% (v/v) penicillin/streptomycin. Medium was exchanged every 2–3 days and cells were

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subcultured weekly in the case of A549 cells and every 10 to 15 days for Calu-3 cell line.

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2.3. Preparation of CS/CRG/TPP nanoparticles

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CS/CRG/TPP nanoparticles were prepared as described previously (Rodrigues, Rosa da

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Costa & Grenha, 2012) based on the polyelectrolyte complexation of CS with CRG and

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additional ionic gelation of chitosan with TPP anions. After an initial optimisation, a final

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theoretical CS/CRG/TPP ratio of 4/1/1 (w/w) was selected to perform the studies (4/1/0 for

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control nanoparticles). Briefly, CS was dissolved in 1% (w/w) acetic acid to obtain a solution

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of 1 mg/mL. CRG and TPP were dissolved in purified water and mixed to obtain a solution

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where the concentration of each polymer is 0.63 mg/mL. The spontaneous formation of

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nanoparticles occurs upon incorporation of 0.8 mL of a solution of CRG/TPP or CRG into 2

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mL of the CS solution, under gentle magnetic stirring at room temperature.

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The association of BSA to the nanoparticles was performed by dissolving the protein in water

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and mixing with a previously prepared mixture of CRG/TPP solution, prior to the addition to

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CS solution, as described above. The amount of BSA associated to each batch was calculated

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to represent a theoretical content of 30% (w/w) of the amount of CS.

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Nanoparticles were concentrated by centrifugation at 16,000 x g on a 10 µL glycerol layer for

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30 min at 15 °C (Heraeus Fresco 17 centrifuge, Thermo scientific, USA). After discarding the

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supernatants, the nanoparticles were resuspended in 200 µl of purified water.

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When no further indication is provided, ‘CS/CRG/TPP nanoparticles’ refer to mass ratios

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2.4. Characterisation of nanoparticles

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2.4.1. Determination of production yield

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The nanoparticle production yield was calculated by gravimetry. Fixed volumes of

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nanoparticle suspensions were centrifuged (16,000 x g, 30 min, 15 °C), and sediments were

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freeze-dried over 24 h at -34 °C, followed by a gradual increase in temperature until 20 °C,

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using a Labconco freeze dryer (Labconco, USA) (n = 3). The yield of nanoparticle production

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(PY) was calculated as follows:

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PY = (Nanoparticle sediment weight/Total solids weight) x 100

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where nanoparticle sediment weight is the weight after freeze-drying and total solids weight

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is the total amount of solids added for nanoparticle formation.

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2.4.2. Morphological observation

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The morphological examination of CS/CRG/TPP nanoparticles was conducted by

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transmission electron microscopy (TEM) (JEM-1011, JEOL, Japan). The samples were

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stained with 2% (w/v) phosphotungstic acid (Carvalho, Grenha, Remunan-Lopez, Alonso &

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Seijo, 2009) and placed on copper grids with carbon films (Ted Pella, USA) for TEM

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observation.

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2.4.3. Physicochemical properties

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Measurements of nanoparticle size and zeta potential were performed on freshly prepared

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samples by photon correlation spectroscopy and laser Doppler anemometry, respectively,

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using a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). For the analysis of particle

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size and determination of the electrophoretic mobility, each sample was diluted to the

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appropriate concentration with ultrapure water and placed in the electrophoretic cell. Each

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analysis was performed at 25 °C. Three batches of each formulation were analysed (n = 3).

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2.4.4. Determination of BSA association capacity

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The amount of non-encapsulated BSA was determined in the supernatant by reverse phase -

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high performance liquid chromatography (RP-HPLC; Agilent 1100 series, Germany). The

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chromatographic conditions were adapted from Umrethia et al. (Umrethia, Kett, Andrews,

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Malcolm & Woolfson, 2010) as follows: Aeris widepore 3.6u XB-C18 300A column 4.6 mm

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i.d.×250 mm length with security guard cartridge (Phenomenex, UK); run temperature 25 ºC;

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gradient flow (0.1% TFA in water (A), 0.1% TFA in acetonitrile (B); A/B from 95:5 to 35:65

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in 10 min) mobile phase, total run time 15 min, UV detection at 280 nm; flow rate 1.0

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mL/min; injection volume 20 µL; BSA retention time 8.8 min. A linear calibration plot for

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BSA in the different solvents (acetic acid solution, PBS pH 7.4 and buffer pH 6.8) was

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obtained over the range 10-120 µg/mL (n = 3). BSA association efficiency (AE) and

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nanoparticle loading capacity (LC) were calculated as follows:

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AE = (Total amount of BSA – Free amount of BSA)/Total amount of BSA × 100

(2)

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LC = (Total amount of BSA – Free amount of BSA)/Nanoparticle weight × 100

(3)

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2.5. Evaluation of nanoparticle stability in the presence of lysozyme

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The stability of CS/CRG/TPP nanoparticles (4/1/0 and 4/1/1) was analysed in presence of

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lysozyme. To do so, nanoparticle suspensions at a concentration of 1 mg/mL were incubated

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with lysozyme (0.2 and 0.8 mg/mL) (Grenha, Seijo & Remuñán-López, 2005) at 37 ºC under

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mild horizontal shaking, for 120 min. At appropriate time intervals (30, 60, 90 and 120 min),

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the size and zeta potential of nanoparticles were measured, as described above (n = 3).

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2.6. Nanoparticle stability upon freeze-drying

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With the aim of developing a pharmaceutically-acceptable dry form of the nanoparticle

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suspensions, CS/CRG/TPP nanoparticles were submitted to a freeze-drying study. Different

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concentrations of nanoparticles (1, 2 and 4 mg/mL) and different cryoprotectants (trehalose,

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lactose, sucrose and glucose) at varied concentrations were tested as variables in the

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optimisation of nanoparticle stabilisation by freeze-drying. To perform the study, 1 mL of

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unloaded nanoparticles was freeze-dried in the presence of 5 or 10% (w/v) of cryoprotectant.

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The freeze-drying process occurred at a pressure of 50–100 Torr and consisted of 24 h of

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primary drying, starting at -35 ºC and gradually increasing to -10 ºC, 24 h at 0 ºC and a final

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step of secondary drying (12–24 h) at 15 ºC (Labconco Corp., USA). After freeze-drying,

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nanoparticle suspensions were reconstituted by adding the volume of water corresponding to

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the initial volume (1 mL), and their size and zeta potential were determined as indicated

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above (n = 3).

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The conditions found to better maintain the original properties of unloaded nanoparticles

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were applied on BSA-loaded nanoparticles. In this case, nanoparticle concentrations of 1 and

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2 mg/mL were freeze-dried with both glucose and sucrose at a concentration of 10% (w/w).

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2.7. Preparation of microencapsulated nanoparticles

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Mannitol was selected as matrix material for the production of microparticles, as was

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previously described (Grenha, Seijo & Remuñán-López, 2005). An aqueous solution of the

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excipient was prepared at a concentration of 1.6% (w/v). Upon centrifugation, CS/CRG/TPP

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nanoparticles were resuspended with the mannitol solution instead of water, in order to obtain

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a mannitol/nanoparticle ratio of 80/20 (w/w). The final solid concentration of the suspensions

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was 2% (w/v).

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Dry powders were obtained by spray-drying the previously prepared suspension using a

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laboratory-scale spray-dryer (Buchi® Mini Spray Dryer, B-290, Buchi, Switzerland).

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The spray-drying conditions were: feed rate of 2.5 mL/min, two fluids external nozzle 0.7

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mm, inlet temperature of 160 ± 2 ºC, outlet temperature of 95 ± 3 ºC. The airflow rate and the

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aspirator were kept constant at 400 Nl/h and 80%, respectively. The spray-dried powders

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were collected and stored in a desiccator at room temperature until use.

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2.8. Characterisation of microencapsulated nanoparticles

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2.8.1. Determination of spray-drying yield

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The spray-drying yield was calculated by gravimetry, comparing the total amount of solids

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initially added for the preparation of microencapsulated nanoparticles (CS + CRG + TPP +

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BSA + mannitol) with the amount of resulting microspheres (n = 3). The following equation

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was used for the calculation:

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Yield (%) = Microspheres weight/Total solids weight × 100

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2.8.2. Morphological and aerodynamic characterisation

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The morphological characterisation of microspheres was performed using a field emission

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scanning electron microscope (FESEM; FESEM Ultra Plus, Zeiss, Germany) at 3 kV of

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voltage, which is equipped with a secondary electron (SE) detector. The dry powders were

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placed onto metal plates and a 5 nm thick gold palladium film was sputter-coated on the

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samples (Q150T S/E/ES Sputter Coater, Quorum Technologies, UK) before viewing.

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The particle size was estimated as the Feret’s diameter (distance between two tangents on

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opposite sides of the particles) and was directly determined with an optical microscope

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(Olympus IX51, Japan). The estimation was based on the mean diameter resulting from the

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evaluation of 300 particles (n = 300).

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Real density was determined using a Helium Pycnometer (Micropycnometer, Quanta

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Chrome, model MPY-2, USA) (n = 3). Apparent tap density was obtained by measuring the

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volume of a known weight of powder in a 10 mL test-tube after mechanical tapping (30

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tap/min, Tecnociencia, Spain). After registration of the initial volume, the test-tube was

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submitted to tapping until constant volume was achieved, according to a previously described

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method (El-Gibaly, 2002) (n = 3).

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The aerodynamic diameter was obtained using a TSI Aerosizer® LD equipped with an

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Aerodisperser® (Amherst Process Instrument Inc.; Amherst, MA, USA) and assuming real

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density as the density parameter for the determination of the aerodynamic diameter.

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2.9. In vitro biocompatibility study

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2.9.1. Evaluation of metabolic activity

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The in vitro cytotoxicity of CS/CRG/TPP nanoparticles, microencapsulated nanoparticles

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(mannitol/nanoparticles = 80/20, w/w), as well as that of the raw materials involved in

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nanoparticle production, was assessed by the metabolic assay thiazolyl blue tetrazolium

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bromide (MTT) test. Two cell lines representative of pulmonary and nasal epithelia (Calu-3

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and A549 cells) were used. A549 cells were seeded at a density of 1×104 cells/well and Calu-

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3 cells at 2×104 cells/well in 96-well plates, in 100 µl of the same medium used for culture in

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cell culture flasks. The cells were grown at 37 °C in a 5% CO2 atmosphere for 24 h before

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use (Grenha et al., 2007).

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Three different concentrations (0.1, 0.5 and 1.0 mg/mL) of nanoparticles were evaluated for

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cytotoxicity over 3 and 24 h. Suspensions of unloaded nanoparticles and microencapsulated

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nanoparticles, BSA loaded nanoparticles and solutions of the individual raw materials (CS,

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CRG and TPP) were evaluated separately for cytotoxicity. Sodium dodecyl sulphate (SDS,

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2%, w/v) was used as a positive control of cell death. Optimal cell seeding density and the

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absence of MTT conversion in the assay medium were confirmed in preliminary experiments.

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All formulations and controls were prepared as solution/suspensions in pre-warmed CCM

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without FBS immediately before application to the cells.

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To initiate the assay, culture medium of cells at 24 h in culture was replaced by 100 µL of

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fresh medium without FBS containing the test samples or controls. After 3 or 24 h of cell

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incubation, samples/controls were removed and 30 µL of the MTT solution (0.5 mg/mL in

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PBS, pH 7.4) were added to each well. After 2 h, any generated formazan crystals were

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solubilised with 50 µL of DMSO. Upon complete solubilisation of the crystals, the

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absorbance of each well was measured by spectrophotometry (Infinite M200, Tecan, Austria)

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at 540 nm and corrected for background absorbance using a wavelength of 650

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nm(Carmichael, DeGraff, Gazdar, MInna & Mitchell, 1987).

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The relative cell viability (%) was calculated as follows:

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Viability (%) = (A – S)/(CM – S)× 100

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where A is the absorbance obtained for each of the concentrations of the test substance, S is

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the absorbance obtained for the 2% SDS and CM is the absorbance obtained for untreated

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cells (incubated with CCM). The latter reading was assumed to correspond to 100% cell

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viability. The assay was performed on three occasions with six replicates at each

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concentration of test substance in each instance.

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2.9.2. Determination of inflammatory response

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The inflammatory response generated by the exposure to nanoparticles and

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microencapsulated nanoparticles was evaluated on Calu-3 cells. To perform the assay, the

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cells were seeded on 96-well plates (2×104 cells/well) and, after 24 h incubation at 37 °C in

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5% CO2 atmosphere, exposed to nanoparticles and microencapsulated nanoparticles at a

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concentration of 1 mg/mL. The exposure to lypopolysacharide (LPS; 10 μg/mL) was used as

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positive control (Alfaro-Moreno et al., 2009; Mura et al., 2011), while cells incubated with

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CCM were used as negative control. All formulations and controls were prepared as

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solutions/suspensions in pre-warmed CCM without FBS immediately before application to

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the cells. After 24 h of exposure, the cell supernatants were collected and centrifuged (10

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min, 16,000 x g, 15 ºC, centrifuge 5804R, Eppendorf, Germany). The levels of IL-6 and IL-8

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existing in Calu-3 cell supernatants were determined by quantitative ELISA using human IL-

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6 and IL-8 Quantikine ELISA kits (R&D Systems, Minneapolis, MN), according to the

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manufacturer’s instructions. The supernatants were appropriately diluted (approximately 10

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times for IL-6 and 50 times for IL-8). The assay was performed on three occasions with two

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replicates of test substance in each instance. The concentration of individual cytokines for

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each sample is expressed as percentage of the control (incubation with CCM) from triplicate

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assays.

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2.9.3. Evaluation of epithelial integrity

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For the studies of epithelial integrity, Calu-3 cells were cultured at an air interface according

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to a previously described methodology (Grainger, Greenwell, Lockley, Martin & Forbes,

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2006). Briefly, 100 µL of Calu-3 cell suspension was seeded at a density of 5×105 cells/cm2

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in Transwell inserts (0.33cm2), with 0.5 mL medium in the basolateral chamber. The cells

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were incubated at 37 ºC, 5% CO2 for 2 days. After this time, the medium was aspirated from

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the apical and basolateral chambers and replaced only in the basolateral chamber, the apical 14 Page 14 of 53

chamber being exposed to the incubator atmosphere. Medium was replaced every 2 days

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thereafter.

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In order to verify the effect of nanoparticles on Calu-3 cell layer integrity, the formulations

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were dispersed in 100 µL FBS-free CCM at a concentration of 1 mg/mL and incubated with

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the cell layers. Transepithelial electrical resistance (TER) was measured using chopstick

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electrodes and an EVOM® volt meter (STX-2 and Evom G, World Precision Instruments,

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UK). TER measurements were taken 30 min before the experiment and at 0.5, 1, 2, 4, 6, 24

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and 48 h after administration. TER values were calculated by subtracting the resistance of

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cell-free culture inserts and correcting for the surface area of the Transwell insert. Cell layers

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incubated with CCM were used as the reference (control), from which TER changes during

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exposure to the samples were calculated. All experiments were performed in triplicate on

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three occasions.

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2.10. Statistical analysis

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For the generality of results, the t-test and the one-way analysis of variance (ANOVA) with

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the pairwise multiple comparison procedures (Dunn’s Method) were performed to compare

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two or multiple groups, respectively.

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The freeze-drying case study determines the effect of different cryoprotectants and

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nanoparticle concentration on nanoparticle stability. Due to the number of observations

318

involved (three to four) in each cell/treatment and to the existence of empty cells, a more

319

complex statistical methodology was not applied, because it would compromise the model

320

assumptions and result in misleading inferences. Therefore, the statistical approach used for

321

this analysis was the one-way ANOVA with Gowes-Howell post-hoc tests, based on sample

322

sizes and variance homogeneity between groups.

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The factorial design (FD) is often used for manipulative experiments, such as that of the

324

metabolic activity (MTT test). In that case, for both cell lines tested (Calu-3 and A549) the

325

effect of sample type (X1) and concentration (X2) on cell viability (Y). Each of the factors (X1

326

and X2) has three levels. In the case of X2 the levels are 0.1, 0.5 and 1.0 mg/mL. For X1 there

327

is a split in two groups: 1) when raw materials are considered the levels of X1 are CS, CRG

328

and TPP; 2) when carrier formulations are studied, the levels are unloaded nanoparticles,

329

BSA-loaded nanoparticles, microencapsulated nanoparticles. For either raw materials or

330

carrier formulations, the objective is to investigate the influence of factors X1 and X2 on the

331

response variable (Y). FD was originally planned with equal number of observations per

332

treatment, but some occurrences related to random loss of observations or to practical

333

constraints, cause inequality of sample sizes. Therefore, for the four multifactor designs

334

present in this study, three were unbalanced with observations in every cell/treatment. For

335

this complex FD, the type III sum of squares (SS) was used, because it is not influenced by

336

the sample size in each treatment. The basic multivariate assumptions to be checked are that

337

the errors are independent, normally distributed and have constant variance. Further analyses

338

with Post hoc tests Hochberg´s GT2 (unbalanced FD) and Tukey, were used to determine

339

which groups differ.

340

The statistical analyses were performed with the software SPSS (version 22.0 for windows)

341

considering a significance level of α = 0.05.

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343

3. Results and discussion

344

CS/CRG/TPP nanoparticles were previously reported by our group in a work that evidenced

345

the ability of TPP to decrease nanoparticle size and improve stability (Rodrigues, Rosa da

346

Costa & Grenha, 2012). In that work, the potential of the carriers for transmucosal drug

347

delivery was suggested. More recently we have observed the ability of these nanocarriers to

16 Page 16 of 53

associate different proteins, demonstrating that the pH of the nanoparticle assembly medium

349

has a strong effect on protein association and the pH of the release medium affects the release

350

profile (unpublished results). The present work is dedicated to the study of these

351

nanoparticles regarding an application in nasal and pulmonary delivery.

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3.1. Characterisation of CS/CRG/TPP nanoparticles

354

After an initial screening of varied CS/CRG/TPP mass ratios, seeking for the formulation that

355

produces nanoparticles with the most suitable characteristics for the objective of

356

transmucosal delivery (Rodrigues, Rosa da Costa & Grenha, 2012), the formulation

357

CS/CRG/TPP = 4/1/1 (w/w) was selected for subsequent studies. The corresponding

358

formulation without TPP (CS/CRG/TPP = 4/1/0, w/w) was used as control in some of the

359

performed studies to show the effect of the inclusion of TPP.

360

Figure 1A depicts the result of the morphological evaluation of BSA-loaded nanoparticles,

361

which evidence a spherical structure similar to both unloaded nanoparticles (Rodrigues, Rosa

362

da Costa & Grenha, 2012) and to polysaccharide-based nanoparticles in general (Goycoolea,

363

Lollo, Remunan-Lopez, Quaglia & Alonso, 2009; Oyarzun-Ampuero, Brea, Loza, Torres &

364

Alonso, 2009; Zorzi, Párraga, Seijo & Sánchez, 2011).

365

As shown in Table 1, the developed BSA-loaded nanoparticles evidence adequate

366

physicochemical properties for the objective of transmucosal delivery, with a size around 300

367

nm and a positive zeta potential (+40 mV). In fact, the size is small enough to permit an

368

intimate contact with epithelial surfaces, which is maximal at 50 – 500 nm (Desai,

369

Labhasetwar, Amidon & Levy, 1996; Jani, Halbert, Langridge & Florence, 1990). In turn, the

370

high positive zeta potential further potentiates the interaction with epithelia, as this is

371

negatively charged and, thus, an electrostatic interaction is enabled. In summary, these

372

characteristics are expected to provide a prolonged retention of nanoparticles close to

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epithelial surfaces, potentiating protein release. Notwithstanding the importance of these

374

physicochemical features, the presence of mucus and the mechanism of mucociliary

375

clearance also need to be considered regarding the success of nasal and pulmonary

376

transmucosal delivery (Dombu & Betbeder, 2013; Marttin, Schipper, Verhoef & Merkus,

377

1998). Nanoparticles described in this work are expected to exhibit bioadhesive properties to

378

some extent, due to the chitosan content. Besides the proper chitosan ability to adhere to

379

biological substrates (Du, Cai & Zhai, 2014; Lehr, Bouwstra, Schacht & Junginger, 1992),

380

chitosan-based nanoparticles have also demonstrated the same capacity in several occasions

381

(Bernkop-Schnürch, Weithaler, Albrecht & Greimel, 2006; Sandri, Bonferoni, Rossi, Ferrari,

382

Boselli & Caramella, 2010). This will possibly improve the carrier residence time close to the

383

epithelia, potentiating drug absorption (Dombu & Betbeder, 2013; Marttin, Schipper, Verhoef

384

& Merkus, 1998). Additionally, nanoparticles have been reported to avoid or delay

385

mucociliary clearance, although this ability is highly dependent on their size and is more

386

relevant for particles below 100 nm (Oberdörster, Oberdörster & Oberdörster, 2005; Sung,

387

Pulliam & Edwards, 2007).

388

It is also observed in the table that the inclusion of TPP in the formulation increases the

389

nanoparticle production yield, while maintaining the encapsulation efficiency, which is

390

around 50%. A great difference is observed in loading capacity, which is of 70% for

391

CS/CRG/TPP = 4/1/0 and approximately 30% for 4/1/1. This difference is explained by the

392

fact that, while both formulations encapsulate a similar amount of protein, the nanoparticles

393

containing TPP display a considerably higher production yield. Therefore, as viewed in Eq. 3,

394

there is the same amount of protein distributed by a higher amount of total solids, thereby

395

resulting in a lower value of loading capacity. Unloaded nanoparticles display somewhat

396

different physicochemical characteristics, 4/1/0 nanoparticles having 491 nm and +78 mV

397

and 4/1/1 nanoparticles displaying a size of 208 nm and +47 mV (data not shown).

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3.2. Evaluation of nanoparticle stability in presence of lysozyme

399

Lysozyme is an enzyme that is present in mucosal surfaces, including the nasal and

400

pulmonary (Konstan, Chen, Sherman, Thomassen, Woodm & Boat, 1981; Muzzarelli, 1997),

401

which exhibits the ability to cleave β-(1-4) glycosidic bonds of chitosan (Muzzarelli, 1997).

402

Therefore, taking into account the high content of chitosan present in the proposed

403

nanoparticles, evaluating their behaviour in presence of this enzyme is important regarding a

404

future interpretation of results, such as those obtained in vivo. To perform the evaluation,

405

nanoparticles were incubated with lysozyme (0.2 and 0.8 mg/mL) at 37 ºC and their size and

406

zeta potential were monitored along time. The enzyme concentration of 0.2 mg/mL was used

407

taking into account indications found in the literature regarding physiological concentrations

408

of the enzyme (Konstan, Chen, Sherman, Thomassen, Woodm & Boat, 1981). Furthermore, a

409

concentration of 0.8 mg/mL was chosen to investigate the nanoparticle behaviour in extreme

410

conditions, as previously reported (Grenha, Seijo & Remuñán-López, 2005).

411

As observed in Figure 2, the formulation of nanoparticles without TPP (CS/CRG) showed a

412

tendency for size decrease along time. When lysozyme was present at a concentration of 0.2

413

mg/mL, the decrease was not significant, as high standard deviations are obtained. However,

414

at 0.8 mg/mL a strong and significant size decrease (p < 0.05) occurs up to 60 min of

415

incubation, although after that a sudden increase in size is observed, inverting the trend. This

416

is probably due to aggregation, as a concomitant increase in the standard deviation of the

417

mean size is observed, indicating a great dispersion of sizes. Zeta potential, in turn, remained

418

stable during the assay (data not shown). Interestingly, the formulation containing TPP was

419

not affected by the presence of the enzyme, no significant size reduction being observed

420

independently of the concentration of lysozyme. This behaviour reveals a somewhat

421

protecting effect attributed by TPP.

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An absence of effect of the enzyme on chitosan-based nanoparticles (also containing TPP)

423

incubated in similar conditions was previously reported in another study (Vila, Sánchez,

424

Tobı́o, Calvo & Alonso, 2002). It is possible that the inclusion of TPP, which acts as cross-

425

linker, prevents or at least modifies chitosan exposure, thus not enabling an easy and rapid

426

enzymatic degradation as is seen for nanoparticles devoid of TPP.

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3.3. Evaluation of nanoparticle stability upon freeze-drying

429

When nanocarriers are formulated as aqueous suspensions, both physical

430

(aggregation/particle fusion) and chemical (hydrolysis of polymer and chemical reactivity)

431

instability phenomena are known to appear at some point (Abdelwahed, Degobert,

432

Stainmesse & Fessi, 2006; Chacón, Molpeceres, Berges, Guzmán & Aberturas, 1999; Kumar,

433

Shafiq & Malhotra, 2012), limiting nanoparticle applications (Sameti et al., 2003; Wu, Zhang

434

& Watanabe, 2011). Aqueous suspensions of CS/CRG/TPP nanoparticles were reported in a

435

previous work to remain stable for up to 9 months, the presence of TPP in the formulation

436

demonstrating to have a significant role in this effect (Rodrigues, Rosa da Costa & Grenha,

437

2012). However, the preference in storing and transporting products in dry state, has led to

438

the decision of developing a lyophilised formulation. Regarding the present work, it is

439

important to recall that a nasal formulation of nanoparticles will involve a suspension, while

440

microencapsulated nanoparticles (dry powders) are proposed for lung delivery. Still, in the

441

latter case there might be a time period between nanoparticle production and

442

microencapsulation where a lyophilized form of nanoparticles will be beneficial for storage.

443

Freeze-drying has been considered an adequate technique to improve the long-term stability

444

of colloidal nanoparticles. Nevertheless, it is a complex process that requires a major

445

optimisation of the inherent conditions. Many parameters of the formulation/process may

446

decide the success of freeze-drying (Abdelwahed, Degobert, Stainmesse & Fessi, 2006). In

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this study, nanoparticles were freeze-dried using different sugars as cryoprotectants, at varied

448

concentrations. The concentration of nanoparticle suspensions was also tested as variable.

449

The direct freeze-drying of nanoparticles (without cryoprotectants) did not permit the

450

preservation of their properties (data not shown), leading to strong aggregation. This effect

451

might be explained by both the high concentration of particles undergoing the freezing step

452

and the mechanical stress induced by ice crystallization (Abdelwahed, Degobert, Stainmesse

453

& Fessi, 2006; Vauthier & Bouchemal, 2009). This observation was reported in a previous

454

work of our group for nanoparticles composed of other polysaccharides (Dionísio, Cordeiro,

455

Remuñán-López, Seijo, Rosa da Costa & Grenha, 2013), as well as in other works available

456

on the literature (Abdelwahed, Degobert, Stainmesse & Fessi, 2006; de la Fuente, Seijo &

457

Alonso, 2008).

458

The purpose of this study was thus examining the effects of type of cryoprotectant (sucrose,

459

trehalose, lactose and glucose), cryoprotectant concentration (5% and 10%, w/w) and

460

nanoparticle concentration (1, 2 and 4 mg/mL) on the nanoparticle characteristics upon

461

reconstitution with water from the corresponding lyophilized powder.

462

Figure 3 displays the variation of nanoparticle size when comparing the obtained value after

463

reconstitution of freeze-dried material (Df) with that initially determined (Di) before the

464

freeze-drying process. As a first approach, unloaded nanoparticles were used. The ANOVA

465

experiment revealed that the concentration of nanoparticle suspensions to be freeze-dried is

466

the sole parameter evidencing a relevant and statistically significant effect on the stability

467

conferred by the cryoprotectants. In this regard, the post-hoc test (Gowes-Howell) shows that

468

using nanoparticle concentrations of 2 mg/mL or 4 mg/mL leads to a higher increase of

469

nanoparticle size (p < 0.05) as compared with 1 mg/mL, which is the concentration

470

permitting the desired maintenance of size after freeze-drying.

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21 Page 21 of 53

Zeta potential values did not evidence alterations after freeze-drying in any of the tested

472

conditions (data not shown). Other works studying the freeze-drying of comparable

473

concentrations of polysaccharide-based nanoparticles have reported good stabilization

474

provided by the same or similar cryoprotectants (de la Fuente, Seijo & Alonso, 2008;

475

Dionísio, Cordeiro, Remuñán-López, Seijo, Rosa da Costa & Grenha, 2013; Hirsjärvi,

476

Peltonen & Hirvonen, 2009).

477

Although not to a statistically significant manner, other interpretations might be suggested by

478

Figure 3. Regarding the performance of the distinct cryoprotectants that were tested, it is

479

important to observe that lactose (a typical excipient used in lung delivery) revealed

480

absolutely inefficient as cryoprotectant agent, not preventing particle aggregation after

481

freeze-drying. This poor protective effect was also observed in a previous work of the group

482

with other polysaccharide-based nanoparticles (Dionísio, Cordeiro, Remuñán-López, Seijo,

483

Rosa da Costa & Grenha, 2013) and was further reported in the literature, being attributed to

484

an ineffective protection during the freezing step (Hirsjärvi, Peltonen & Hirvonen, 2009).

485

Sucrose, glucose and trehalose (in this order) are suggested to have better ability as

486

cryoprotectants, as their presence provided a more adequate maintenance of the initial

487

characteristics of nanoparticles, at least under certain conditions. Sucrose maintained the

488

exact characteristics of nanoparticles when used at 10% (with the already mentioned

489

exception of the 4 mg/mL concentration of nanoparticles) or at 5% when nanoparticles are

490

freeze-dried at the lower concentration tested. A very satisfactory cryoprotective effect

491

provided by both glucose and sucrose was previously reported by our group regarding the

492

stabilisation of two formulations of pullulan-based nanoparticles (sulfated pullulan/chitosan

493

and carrageenan/aminated pullulan) (Dionísio, Cordeiro, Remuñán-López, Seijo, Rosa da

494

Costa & Grenha, 2013). The concentration of the cryoprotectant is also an important

495

parameter to optimise. In this regard, a content of 5% was generally found to be enough to

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22 Page 22 of 53

stabilise nanoparticles freeze-dried at a concentration of 1 mg/mL, while an increase to 10%

497

of cryoprotectant is needed when the nanoparticle concentration increases to 2 mg/mL.

498

Considering the results obtained with unloaded nanoparticles, BSA-loaded nanoparticles at

499

the concentration of 1 and 2 mg/mL were freeze-dried with 10% of both glucose and sucrose.

500

The physicochemical characteristics of the nanoparticles were still maintained (data not

501

shown). However, it was previously referred that the use of glucose as cryoprotectant of

502

protein formulations should be cautious because of its reducing character (Li, Patapoff,

503

Overcashier, Hsu, Nguyen & Borchardt, 1996; Wang, 2000). Nevertheless, the freeze-drying

504

of protein nanocarriers in presence of glucose has been reported as efficient, considering both

505

the physicochemical stability of the carrier and the maintenance of protein properties

506

(Freixeiro et al., 2013). In any case, a comprehensive review on the subject suggests that any

507

evaluation should be performed on a case-by-case basis (Wang, 2000).

508

This study evidences the suitability of using freeze-drying to obtain nanoparticles in a dry

509

form, but it reinforces the importance of carefully selecting and optimising the conditions of

510

the freeze-drying process in order to achieve an optimised protective effect.

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512

3.4. Microencapsulation of nanoparticles

513

The success of inhalable formulations is primarily dependent on their aerodynamic

514

properties, as these influence both the dispersion and sedimentation pattern of the carriers

515

(Sung, Pulliam & Edwards, 2007). When a systemic action is intended, the drug carriers

516

should be small enough to pass through the mouth, throat and conducting airways and reach

517

the deep lung, but not so small that they fail to deposit and are breathed out again. In this

518

context, it has been considered that an aerodynamic diameter between 1 and 5 µm is

519

necessary (Bosquillon, Lombry, Préat & Vanbever, 2001), which in some cases is even

520

narrowed to 2-3 µm (Oberdörster, Oberdörster & Oberdörster, 2005). A specific conjugation

23 Page 23 of 53

between size and density of the carriers is, thus, necessary. The pulmonary administration of

522

nanoparticles is severely hindered by their low inertia, which makes alveolar deposition

523

practically impossible, mainly resulting in the exhalation of the carriers (Sung, Pulliam &

524

Edwards, 2007). The spray-drying of CS-based nanoparticles was first reported by Grenha et

525

al. (2005) as a strategy to endow the nanoparticles with the ability to reach the deep lung.

526

This strategy was later on followed by other groups (Li, Sun, Gong, Liu, Zuo & Zhang, 2010;

527

Pourshahab, Gilani, Moazeni, Eslahi, Fazeli & Jamalifar, 2011). In the present work, the

528

microencapsulation of CS/CRG/TPP nanoparticles was approached to obtain a drug delivery

529

system that permitted efficient administration of the developed nanocarriers aimed at the

530

systemic pulmonary delivery of proteins. Mannitol was selected to compose the matrix of

531

microparticles, as it has demonstrated to simply act as carrier of nanoparticles, providing their

532

release upon reaching the alveoli (Grenha, Seijo & Remuñán-López, 2005).

533

Microencapsulated nanoparticles were produced by spray-drying a suspension of

534

mannitol/nanoparticles (80/20, w/w), evidencing a spherical shape and a slightly corrugated

535

surface, as can be observed in Figure 1B. The nanoparticle carriers were determined to have a

536

Feret diameter of 2.3 ± 0.6 µm, a real density of 1.44 ± 0.01 g/cm3 and a tap density of 0.42 ±

537

0.04 g/cm3. The aerodynamic diameter was determined to be 1.80 ± 0.11 µm. Comparable

538

results were reported for microencapsulated CS/TPP nanoparticles (Grenha, Seijo &

539

Remuñán-López, 2005), a system that already demonstrated its efficacy in vivo for the

540

administration of insulin (Al-Qadi, Grenha, Carrión-Recio, Seijo & Remuñán-López, 2012).

541

Good indications are thus provided on the capacity of the developed system to act as carrier

542

of nanoparticles to the lung with the aim of a systemic delivery of proteins. Nevertheless,

543

using this approach of microencapsulating the nanoparticles could possibly pose an extra

544

limitation, regarding the high proportion of excipients that need to be used and, consequently,

545

the inherent lower loading. Taking into account that administrations of around 160 – 180 mg

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24 Page 24 of 53

of dry powder have been reported (Hoppentocht, Hagedoorn, Frijlink & de Boer, 2014;

547

Labiris, Holbrook, Chrystyn, Macleod & Newhouse, 1999) and considering a protein loading

548

of 27% in nanoparticles and the 20% amount of nanoparticles in the final microparticle

549

formulation, 160 mg would carry approximately 9 mg of protein. This amount is considered

550

suitable for the purpose of obtaining a therapeutic effect and will possible not compromise

551

the success of the formulation, which will obviously be dependent on several other

552

parameters.

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553

3.5. In vitro biocompatibility study

555

Evaluating the biocompatibility of drug delivery formulations is nowadays a mandatory

556

aspect to address (Gaspar & Duncan, 2009). While an accurate determination of the toxicity

557

of a formulation can only be determined in vivo, a variety of in vitro toxicological assays,

558

performed in adequately selected cell lines, might provide very useful information and are

559

widely accepted as first indicators (ISO, 2009; Rodrigues, Dionísio, Remuñán-López &

560

Grenha, 2012). Current international guidelines require the evaluation to be contextualised

561

with a specific route of administration and dose of the material (Gaspar & Duncan, 2009). It

562

is also referred that polymers and polymer-based carriers should be regarded as different

563

entities and, thus, evaluated separately (Aillon, Xie, El-Gendy, Berkland & Forrest, 2009;

564

Gaspar & Duncan, 2009; Williams, 2008). This is based on the assumption that the proper

565

carrier structure, among other parameters, might affect the final toxicological behaviour

566

(Aillon, Xie, El-Gendy, Berkland & Forrest, 2009; Gaspar & Duncan, 2009).

567

To address the above mentioned issues, the evaluation of the biocompatibility of

568

CS/CRG/TPP nanoparticles and corresponding raw materials, was performed in this study by

569

means of three different assays, 1) the metabolic activity (MTT), 2) the inflammatory

570

response (detection of IL-6 and IL-8) and 3) the epithelial integrity (measurement of TER).

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Calu-3 and A549 were the selected cell lines, as they are representative of the respiratory

572

epithelia. Calu-3 is an immortalised cell line obtained from lung adenocarcinoma and has

573

been extensively used in the study of formulations designed for either nasal or pulmonary

574

drug delivery, as it is considered a model of the epithelium of both regions (Casettari,

575

Vllasaliu, Mantovani, Howdle, Stolnik & Illum, 2010; Grainger, Greenwell, Lockley, Martin

576

& Forbes, 2006; Zhu, Chidekel & Shaffer, 2010). Importantly, Calu-3 cells form tight

577

junctions in vitro (Winton et al., 1998) and, under suitable culture conditions, exhibit in vivo-

578

like barrier properties (Grainger, Greenwell, Lockley, Martin & Forbes, 2006). A549 is a cell

579

line representative of the alveolar epithelium, obtained from human alveolar adenocarcinoma,

580

showing no ability to form functional tight junctions in culture (Forbes & Ehrhardt, 2005;

581

Foster, Yazdanian & Audus, 2001; Winton et al., 1998). Consequently, it cannot be used in

582

studies of the epithelial barrier integrity, although this does not prevent their use in

583

cytotoxicity studies.

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3.5.1. Evaluation of metabolic activity (MTT assay)

586

The MTT assay provides an evaluation of the cell metabolic activity upon exposure to the

587

samples and has been used very frequently in the assessment of drug delivery carriers

588

(Rodrigues, Dionísio, Remuñán-López & Grenha, 2012). It evaluates the ability of the cells

589

to reduce the MTT reagent to tetrazolium salts, an action that is dependent on mitochondrial

590

metabolism. A reduction of cellular metabolic activity is generally accepted as an early

591

indicator of cellular damage (Scherließ, 2011).

592

In this work, A549 and Calu-3 cells were cultured and exposed to different concentrations of

593

nanoparticles and raw materials for both a short (3h) and a more prolonged period (24h). The

594

exposure for 3h did not lead to significant decrease in cell viability, with values remaining

595

close to 90-100% in all cases. The only exception occurred for A549 cells exposed to

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26 Page 26 of 53

chitosan (data not shown), an effect commented later. In contrast, the prolongation of

597

exposure up to 24h induces some variability on cell viability. In this manner, for the contact

598

time of 24h a factorial design was approached to examine the effect of sample type (raw

599

materials/carrier formulations; X1) and concentration (X2) on cell viability (Y), for each cell

600

line. The two factors (X1, X2) of the experimental design have three levels each, resulting in

601

nine factor/level combinations.

602

Cell viability results obtained upon 24h exposure are depicted in Figures 4 (Calu-3 cells) and

603

5 (A549 cells). For Calu-3 cells, an unbalanced FD was used in the group of raw materials

604

and formulations. For the raw materials (Figure 4A), a significant effect (p < 0.05) was

605

detected for each of the main factors (X1 and X2) and their interaction (X1*X2). The most

606

relevant observation is that TPP induced a greater decrease of cell viability as compared to

607

CS and CRG (p < 0.05).

608

With respect to the formulations, the factorial experiment reveals that only X1 (type of

609

materials) and the two-way interaction X1*X2 are statistically significant (p < 0.05). Although

610

the lowest viability induced by raw materials was observed for the highest concentration of

611

TPP (1.0 mg/mL, Figure 4A), cell viability upon contact with CS/CRG/TPP nanoparticles

612

was above 95% in all instances (Figure 4B), no concentration-dependent effect being

613

observed. In this manner, as a cell viability of 70% is the threshold beyond which a cytotoxic

614

effect is considered to occur according to ISO 10993-5 (ISO, 2009), the observed effects are

615

devoid of physiological relevance. This indicates that Calu-3 cells do not evidence a

616

particular sensitivity to any of the tested samples, either raw materials or nanoparticle-based

617

formulations, at the tested concentrations.

618

In A549 cells, a balanced FD of the raw materials showed a significant effect for both main

619

factors (X1 and X2) and their interaction (X1*X2). Among raw materials only CRG and TPP

620

show a significant difference between the concentration levels (p < 0.05), cell viability

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27 Page 27 of 53

decreasing with increasing concentrations, particularly at the concentration of 1 mg/mL

622

(Figure 5A). Nevertheless, cell viabilities were higher than 70% in all conditions tested for

623

both polymers. The most deleterious effect was observed for chitosan (p < 0.05), which

624

induced high cytotoxicity, as was also detected upon 3h, even at the lower concentration of

625

0.1 mg/mL.

626

The different response of both cell lines to CS exposure (in Calu-3 cells no viability decrease

627

was observed) is most probably related with the different structures evidenced by Calu-3 and

628

A549 cells in culture and with their sensitivity to pH alterations. In fact, while Calu-3 cells

629

are rounder, A549 cells grow according to a more extended profile, as can be easily seen in

630

ATCC webpage (Collection, 2012). Chitosan used in this work is a base and, therefore, is

631

dissolved in acetic acid (pH around 3.2) to assemble the nanoparticles. The same solution was

632

used in the assay, although it was previously diluted with CCM. Upon addition to the cell

633

layer, it was observed on the microscope that A549 cells begin to detach, an effect not

634

perceived for Calu-3 cells. Therefore, the cells are no longer attached to the plate and are

635

easily removed during the washing steps even before the MTT reagent is added, not

636

permitting a reliable measure of the metabolic activity. This indicates that these results should

637

be taken with precaution. Importantly, although there are not many studies testing chitosan

638

solutions in A549 cells, the literature reports an absence of overt toxicity of the polymer in

639

this cell line at concentrations similar to those used in this study, although a different chitosan

640

polymer (regarding molecular weight and salt) was used (Huang, Khor & Lim, 2004).

641

The behaviour of A549 cells regarding the tested drug delivery systems was generally similar

642

to that of Calu-3 cells, evidencing a very favourable profile. The statistical details of the FD

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showed that both main effects (X1 and X2) and their interaction (X1*X2) are significant (p