Chitosan acetylation degree influences the physical properties of

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Biological and Medical Applications of Materials and Interfaces

Chitosan acetylation degree influences the physical properties of polysaccharide nanoparticles: implication for the innate immune cells response Franco Furlani, Pasquale Sacco, Eva Decleva, Renzo Menegazzi, Ivan Donati, Sergio Paoletti, and Eleonora Marsich ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21791 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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ACS Applied Materials & Interfaces

Chitosan Acetylation Degree Influences the Physical Properties of Polysaccharide Nanoparticles: Implication for the Innate Immune Cells Response

Franco Furlani 1, ‡, Pasquale Sacco 1, ‡, *, Eva Decleva 1, Renzo Menegazzi 1, Ivan Donati 1, Sergio Paoletti 1 and Eleonora Marsich 2

1 Department

2 Department

of Life Sciences, University of Trieste, Via L. Giorgieri 5, I-34127 Trieste, Italy

of Medicine, Surgery and Health Sciences, University of Trieste, Piazza dell’Ospitale 1, I34129 Trieste, Italy

‡These authors contributed equally

* Corresponding author E-mail: [email protected] Phone: +39-040-5588733

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Abstract The aim of the present contribution is twofold, as it reports: i) on the role played by chitosan acetylation degree for the stability of nanoparticles formed with hyaluronan (NPs); ii) on the effect of the interaction of such NPs with immune cells. Chitosans with similar viscosity-average molecular weight, 𝑀𝑣, (i.e. 200 000) and different fraction of acetylated units (FA), together with a low molecular weight hyaluronan were chosen for developing a selected library of formulations via electrostatic complex coacervation. Resulting NPs were analyzed in terms of size, polydispersity, surface charge and stability in physiological-mimicked media by Dynamic Light Scattering (DLS). Only medium acetylated chitosan (FA = 0.16) guaranteed the stability of nanoparticles. To explore the effect of NPs interaction with immune cells, the release of pro-inflammatory cytokines and reactive oxygen species (ROS) production by human macrophages and neutrophils, respectively, were evaluated. Strikingly, a structure-function relationship emerged, showing that NPs made of chitosans with FA = 0.02, 0.25, 0.46 and 0.63 manifested a pro-inflammatory activity, linked to the instability of the system. Conversely, NPs made of chitosan with FA = 0.16 neither modified the functional response of macrophages nor that of neutrophils. Of note, such NPs were found to possess additional properties potentially advantageous in applications as means of delivery of therapeutics to target inflamed sites, namely: (i) they are devoid of cytotoxic effects, (ii) they avoid engulfment during the early stage of interaction with macrophages and, (iii) they are mucoadhesive, thereby providing for site-specificity and long-residence effects.

Keywords: chitosan acetylation degree; nanoparticles; stability; macrophages; neutrophils; inflammation.


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1. Introduction Chitosans are biopolymers deriving from the partial/total deacetylation of chitin, the second most abundant polysaccharide on earth. From the structural point of view, such polymers are composed of two building β-1→4 linked sugars, i.e. glucosamine, GlcNH2 (“deacetylated”, D unit) and N-acetyl-glucosamine, GlcNAc (“acetylated”, A unit) randomly distributed along the polysaccharide chain. In contrast to chitin, all chitosans are soluble in acidic aqueous solutions following the protonation of primary amino groups of D sugars in the C2 position. The fraction of A units, FA, determines the acetylation degree of chitosan. The degree of acetylation influences important parameters, such as charge density, crystallinity, solubility and susceptibility to enzymatic degradation.1 Chitosan (CH) is low toxic, non-immunogenic and biodegradable.2,3 This biopolymer is extremely versatile, and can be used to develop different systems as films, scaffolds, gels, micro/nano-fibers, and micro/nano-particles.4–9 Due to its undeniably interesting properties, chitosan is widely used for biomedical and pharmaceutical applications for the realization of systems able to encapsulate and deliver therapeutic molecules.6,7,9–15 Complex coacervation of chitosan with negatively charged polysaccharides as hyaluronan or alginate is a simple method for synthetizing ensembles of colloids to be used as carriers in very different fields of application, spanning from the delivery of genes to that of proteins.16,17 Hyaluronic acid (HA) is of particular interest due to its ability to interact with its membrane receptor CD44,18 known to be overexpressed in certain cancers, such as those affecting breast 19 and liver,20 thereby cueing antineoplastic drugs for a selective targeting. Chitosan/hyaluronan (CH/HA) nanoparticles (NPs) have been designed to encapsulate heparin for pulmonary delivery applications,21 and proved to be suitable for the encapsulation of growth factors as VEGF and PDGF-BB.22 Moreover, such NPs have been used to encapsulate plasmids with high efficiency.12 Very little is known about the influence of FA on the stability of CH/HA NPs and on the biological implications, since (i) most authors use commercial chitosans with very similar physicalACS Paragon Plus Environment

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chemical properties, i.e. FA < 0.2 and medium-to-high molecular weight, and (ii) some of them do not even report any information about the acetylation degree of chitosans. An exception is represented by the work of Delair and co-authors where an in depth characterization of chitosans is reported in relation to the formation and stability of polyelectrolyte complexes using hyaluronan as the polyanion.23 In the same work, the authors claimed that the stability of such coacervates, prepared in medium with osmolarity and pH close to physiological values, could be achieved using metallic ions (namely Zn2+). When foreign particles, such as NPs, are administered into the body, activation of the innate immune system occurs: neutrophils act as the first defense line, producing pro-inflammatory reactive oxygen species (ROS, e.g. H2O2) and releasing granular components (e.g. myeloperoxidase); in the meantime, macrophages secrete cytokines as Tumor Necrosis Factor-α (TNF-α) which may contribute to tissue damage.24 Shape and dimension of nano-systems - together with rigidity - are cues that may influence the immunological response and internalization both in vitro and in vivo.25–27 Owing to the complexity of the immune response elicited by nanomaterials,28 a first necessary step to devise an effective drug delivery system is to evaluate the in vitro response of innate immunity cells. The goal of this work is to investigate the influence of chitosan acetylation degree on the NPs stability and on their capability to induce immunological responses. On the basis of a previous contribution by our group,29 a selected library of formulations were synthetized by adopting an experimental protocol requiring mild operating conditions. The latter protocol requires the additional presence of small amounts of the polyanionic species tripolyphosphate, TPP. The resulting NPs were characterized from a physical-chemical point of view and their stability in conditions mimicking physiological media was investigated. Two cellular models, namely human macrophages and neutrophils, were selected to study the influence of NPs on the viability and functional responses of these phagocytes. Overall, we herein report that NPs integrity is a pivotal


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prerequisite for cellular response. Finally, the ability to interact with or to cross the cellular membrane, as well as NPs muco-adhesive properties, was investigated.

2. Materials and methods 2.1 Materials. Hydrochloride chitosans (CH) with similar molecular weight (viscosityaverage molecular weight, 𝑀𝑣, ~ 200 000) and different fraction of acetylated units, FA, (determined by 1H-NMR) were kindly provided by Novamatrix/FMC Biopolymer (Sandvika, Norway) and by the late Prof. Kjell Morten Vårum (NTNU, Trondheim, Norway). Hydrochloride chitosan sample with FA = 0.02 was obtained via heterogeneous deacetylation

30

starting from a chitosan template

with an original FA = 0.14. Hydrochloride chitosan sample with FA = 0.25 was obtained via re-Nacetylation

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starting from a chitosan template with an original FA = 0.16. The characteristics of

CH are presented in Table S2. Endotoxins content in chitosan samples was determined by kinetic turbidimetric LAL test according to Ph. Eur. 7.0, 2.6.14, Method C. Sodium hyaluronate (HA), ([𝜂] = 270 mL/g, 𝑀𝑣 = 90 000, Bioibérica S.A.) was kindly provided by Sigea Srl (Trieste, Italy). Sodium

tripolyphosphate

pentabasic

(TPP

≥

98%),

N-(3-dimethylaminopropyl)-N'-

ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Phosphate Buffered Saline (PBS), Percoll, bovine serum albumin (Cohn fraction V BSA, ≥ 96% cell culture-tested), dihydrorhodamine 123 (DHR), horseradish peroxidase (HRP), type VI, fibrinogen (FBG), from human plasma, phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS), NaHCO3, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), ethanol, mucin (from porcine stomach, type III, bound sialic acid 0.5-1.5%, partially purified), penicillin, streptomycin and glutamine were from Sigma-Aldrich Co. (St.Louis, MO). RPMI (Roswell Park Memorial Institute) 1640 and Fetal Bovin Serum (FBS) were from EuroClone (Italy). High-purity Trypan Blue (TB, Color Index 23850) - obtained from Merck KgaA (Darmstadt, Germany) - was dissolved in distilled water at 5 mg/mL and filtered through a Millipore filter to remove non-solubilized material. All other reagents were from Sigma-Aldrich. All reagents and chemicals were of high purity grade. Deionized (Milli-


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Q) water was used in all experiments, except for the solutions used in the biological assays, which were prepared in endotoxin-free water or physiologic saline (0.9% w/v NaCl) for clinical use. 2.2 Synthesis of nanoparticles (NPs). The synthesis of NPs was performed according to a previously reported procedure.29 Briefly, polymers and TPP were solubilized in deionized water at a concentration equal to 0.6 mg/mL for chitosan, 1.25 mg/mL for sodium hyaluronate and 0.5 mg/mL for TPP. After complete solubilization, polymer solutions were filtered through 0.22 μm filters (Biosigma, Italy) and stored at room temperature until use. 150 μL of TPP solution were then added dropwise to 3 mL of hyaluronate solution under stirring. 500 μL of the resulting HA/TPP solution were added dropwise to 1 mL of chitosan solutions placed in 5 mL beakers under stirring to allow for the formation of NPs. The final weight ratio between CH and HA was 1:1 w/w. Solutions were kept under stirring for 10 min and left at rest for 20 min allowing nanoparticles to stabilize. All the relevant concentrations pertaining to the different (final) NPs components in the cases of chitosans having different values of FA have been reported in Table S1. 2.3 Physical-chemical characterization of NPs. 2.3.1 NPs Size, Surface Charge and Homogeneity. Formulations were analyzed by means of Dynamic Light Scattering (DLS) on a Zetasizer Nano ZS with 173° detection optics (Malvern Instruments) to evaluate their size (hydrodynamic diameter), PolyDispersity Index (PDI) and surface charge (𝜁-potential). Each formulation was analyzed using disposable cuvettes at least in triplicate at T = 25 °C after dilution 1:10 v/v in deionized filtered water. The size was expressed as the Z-average hydrodynamic diameter, obtained by a cumulative analysis of the correlation function using the viscosity and refractive index of water in the calculations. 𝜁-potential was determined via Laser Doppler Velocimetry (LDV) technique. 2.3.2 Stability of nanoparticles. The stability of NPs was evaluated by DLS using PBS as buffer.22 The composition of PBS is: NaCl 137 mM, KCl 2.7 mM, phosphate buffer 10 mM with final ionic strength (I) of 168 mM and pH 7.4. Simulated Body Fluid (SBF) was used as additional ACS Paragon Plus Environment

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medium to study NPs stability. The composition of SBF was: NaCl 136.8 mM, NaHCO3 4.2 mM, KCl 3 mM, K2HPO4 1 mM, MgCl2.6H2O 1.5 mM, CaCl2.2H2O 2.5 mM, Na2SO4 0.5 mM, Tris-base 50 mM with final ionic strength (I) of 184 mM and pH 7.4. HEPES buffer (10 mM)/NaCl (150 mM) was finally used as medium to check NPs stability. Resulting formulations were analyzed in triplicate by means of DLS after dilution 1:10 v/v in the appropriate media. The considered parameters to assess the stability of NPs were: (i) DLS size quality report, (ii) size and volume distribution curves and (iii) PDI values. 2.4. In vitro assays with macrophages. Macrophage-like cells obtained from the differentiation of the human pro-monocytic cell line U937 (Sigma) were used as the cellular model.32 Undifferentiated U937 were cultured in suspension in RPMI-1640, supplemented with 10% v/v fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM Lglutamine in an atmosphere with 5% CO2 at 37 °C. To induce differentiation, 150 000 cells/well in 400 μL of RPMI were plated in 24-well tissue culture plates, and 100 μL/well of PMA diluted in RPMI (15 ng/mL final concentration) were added. After three days of incubation at 37 °C, supernatants were removed and replaced with the same volume of fresh medium. Macrophages were let to restore for two days. Cell medium was then discarded, replaced with 450 μL/well of fresh medium and finally 50 μL of water (in the case of control), NPs and polymer solutions added to each well. In each experiment, macrophages exposed to 1 ng/mL LPS were considered as positive controls. After three hours of incubation, supernatants were recovered and centrifuged at 13 000 x g for 10 min to remove any cellular debris. NPs used in these experiments were prepared as described in paragraph 2.2 using sterile glassware and instruments. All solutions were prepared using water for injection (Eurospital SpATrieste). Solutions were further sterilized by filtration using filters with a pore diameter equal to 0.22 μm. 2.4.1 Viability test. The viability of adherent U937 macrophages was assessed using the


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Neutral red assay.33 Briefly, neutral red powder was dissolved in water (5 mg/mL final concentration), centrifuged at 16 000 x g for 10 min to remove any insoluble residue, and stored in dark conditions until use. Neutral red was diluted in “warm” PBS (T = 37 °C) to obtain a final concentration of 100 μg/mL. 400 μL of the dye were added to each well. The plate was incubated at 37 °C for 20-30 min, then the dye was removed; each well was washed once with 500 μL of warm PBS and 400 μL of a 50% v/v ethanol/water plus 1% v/v acetic acid solution were then added to each well. Finally, a 150 μL-volume was transferred from each well into a 96 multi-well plate and the absorbance measured at 540 nm using a plate reader spectrophotometer (Infinite 200Pro NanoQuant, Tecan Trading AG, Switzerland). 2.4.2 Cytokine production. ELISA tests (Invitrogen Corporation, Thermo Fischer, USA) were carried out according to manufacturer’s protocol for the quantification of TNF- (tumor necrosis factor-) secreted by U937 macrophages upon NPs treatment. 2.4.3 Membrane association and internalization. To evaluate membrane association and cellular internalization of the NPs, fluoresceinamine isomer I-labeled-HA was used for the synthesis of NPs. HA was labeled according to a procedure reported elsewhere.29 The association of fluorescence-labeled NPs with macrophage membrane and their internalization were determined by means of flow cytometry according to the method described by Busetto et al.,34 with minor modifications. Cells were differentiated as described in paragraph 2.4. and, after 3 or 24 h of incubation with the NPs, the supernatants were discarded and the cells washed with PBS. Cells were then gently scraped using a plastic spatula, re-suspended in ice-cold PBS supplemented with CaCl2 1 mM and centrifuged for 5 min at 125 x g. Supernatants were discarded and pellets resuspended in ice-cold PBS supplemented with CaCl2 1 mM, yielding a final concentration of 2 x 106 cells/mL. Cells were put on ice prior analyses. Flow cytometry was performed with a FACSCalibur (BectonDickinson) equipped with an air-cooled 15 mW argon-ion laser, operating at 488 nm. Immediately before the analysis, 250 μL of each sample were diluted with an equal volume of ice-cold 0.1 M citrate buffer, pH 4. Control cells were analyzed as they were to determine the ACS Paragon Plus Environment

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autofluorescence, and after addition of 50 μL of NPs to determine NPs association. To distinguish between cell association and internalization, each sample was analyzed after 1 min from the addition of TB (75 μg/mL final concentration). Fluoresceinamine isomer I fluorescence (FL1, green) was collected using a 530 (± 30) nm bandpass filter, instead red fluorescence emitted after quenching by TB (FL3) was collected by using a 650 (± 13) nm bandpass filter. Data were collected using a linear amplification for FSC and SSC, and a logarithmic amplification for FL1 and FL3. For each sample 10 000 events were collected and analyzed using the CellQuest software from Becton Dickinson. Macrophages population was identified by combined measurement of FSC and SSC and gated in R1 region. The percentage distribution of macrophage subsets was calculated from dot plot analyses (FL-1 vs. FL-3) of R1-gated events. 2.5 In Vitro Assays with Neutrophils. 2.5.1 Neutrophil Isolation. Institutional ethics committee approval was obtained and written informed consent was signed by healthy volunteers from which venous blood was withdrawn. Neutrophils were isolated by a discontinuous Percoll gradient centrifugation, as previously described,35 and suspended in PBS solution, pH 7.4, containing 5 mM glucose and 0.2% w/v BSA (PBS-BSA). 2.5.2 Preparation of FBG-Coated Surfaces. Flat-bottom poly(styrene) wells (F16 MaxiSorp Nunc-Immuno Modules or F16 Black MaxiSorp Fluoronunc Cert, Thermo Fisher Scientific, Roskilde, Denmark) were coated with FBG as described elsewhere.36 Briefly, 50 μL of FBG solution (400 μg/mL in PBS) were put in each well, and the plate was left at 37 °C for 1-2 h in a humidified chamber. Just before use, the wells were rinsed three times with PBS. 2.5.3 Evaluation of H2O2 production. Neutrophils H2O2 generation was assessed using the conversion of non-fluorescent DHR into fluorescent rhodamine-123 in the presence of HRP.37–39 Neutrophils (1.25 × 106 cells/mL in PBS-BSA) were incubated with 40 M DHR for 30 min at 37 °C in a shaking water bath, in the dark. Five to ten min before starting the assay, the cell suspension


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was supplemented with 1 mM CaCl2 and 1 mM MgCl2. Then, 60 μL aliquots of this suspension were dispensed into FBG-coated black wells containing or not the NPs (chitosan with FA = 0.16, 80 µg/mL final concentration), and HRP (1 g/mL final concentration), in a total volume of 0.15 mL PBS-BSA supplemented with 1 mM CaCl2 and 1 mM MgCl2 (Ca2+/Mg2+ PBS-BSA). The plate was incubated at 37 °C in the dark and at the desired times readings were taken with a microplate fluorescence reader (Tecan Infinite F200; Tecan Austria GmbH, Grödig, Austria) at 485 nm (λex) and 535 nm (λem). 2.5.4 Evaluation of granular component release. The effect of NPs on neutrophil degranulation was assayed following the release of myeloperoxidase (MPO), a widely used marker of neutrophil primary granules.40–42 Neutrophils (1.0 × 106 cells/mL in PBS-BSA) were pre-warmed in suspension for 10 min at 37 °C. Five min before starting the assay, the cell suspension was supplemented with 1 mM CaCl2 and 1 mM MgCl2. Then, 150 μL aliquots of this suspension were dispensed into quadruplicate FBG-coated wells containing or not the NPs (chitosan with FA = 0.16, 80 µg/mL final concentration) in a total volume of 200 μL. After 60 or 90 min of incubation at 37 °C, the plates were centrifuged at 200 x g and the supernatants were collected. As a positive control, the MPO released in the supernatant of neutrophils stimulated with the known secretagogue NFormyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) (Sigma-Aldrich) in the presence of cytochalasin D (Sigma-Aldrich) (final concentrations, 5 x 10-7 M and 2.5 µg/mL, respectively) was assayed in a separate plate after 20 min of incubation at 37 °C. Released MPO was measured by using 3,3,5,5-tetramethylbenzidine (TMB) (Sigma-Aldrich) as substrate following the method described by Menegazzi et al. with some modifications.42 Briefly, 75 µL of 20 mM acetate buffer (pH 5.5) containing 2 mM TMB and 0.1% (w/v) cetyltrimethylammonium bromide (SigmaAldrich) were deposited into wells of a separate plate. Then, 75 µL of either supernatant fluid or 20fold diluted neutrophil lysate were added to each well. The peroxidatic reaction was started with H2O2 (0.3 mM final concentration) and blocked after 3-5 min with 100 μL of 2 N H2SO4. Absorbance was then read at 405 nm. The amount of released MPO was expressed as percentage of ACS Paragon Plus Environment

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the total MPO activity as measured in neutrophil lysates. 2.5.5 Measurement of Neutrophil Adhesion. The number of neutrophils adherent to FBG was assessed by quantifying myeloperoxidase (MPO) activity as described in the paper by Menegazzi et al.35 Adhesion tests were conducted in FBG-coated transparent wells to monitor cell morphology by light microscopy. They were run in parallel to those evaluating H2O2 production, and in the same experimental conditions except for the omission of DHR and HRP. 2.6 Evaluation of NPs muco-adhesiveness. The muco-adhesive properties of NPs were evaluated in vitro by assessing the interaction between NPs and mucin.43 Mucin was purified via dialysis against deionized water prior to use. The solution was thereafter filtered through 8 μm Millipore filters and freeze-dried. Freeze-dried mucin was solubilized in deionized water at 2 mg/mL prior to use. NPs (80 μg/mL final concentration) were added (1:10 v/v, 1 mL final volume) to solutions of mucin at different concentrations (0.1, 0.2, and 0.5 mg/mL). Resulting mixtures, which had a final pH ~ 5.5, were re-suspended under vigorous stirring and analyzed by DLS. The change in NPs size distribution was used as a parameter to evaluate aggregation phenomena. 2.7 Statistical analysis. Statistical analysis was performed using the Unpaired Student’s ttest, using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA). p-values less than 0.05 were considered statistically significant.

3. Results and discussion 3.1 Physical-chemical characterization of NPs and stability studies. To study the physical properties of nanoparticles (i.e. size, polydispersity and surface charge) synthetized using chitosans with different FA, DLS analyses were carried out. 3.1.1. DLS characterization of NPs prepared with different FA chitosans. At first, we decided to perform DLS analyses on NPs in water, prepared at their unbuffered pH value. The results are reported in Table 1.


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FA

medium

pH

hydrodynamic diameter (nm)

PDI

0.02

water

4.6

513 ± 13

0.24 ± 0.02

37 ± 5

no aggregation

0.16

water

5.6

207 ± 12

0.21 ± 0.01

32 ± 1

no aggregation

0.25

water

5.8

348 ± 11

0.24 ± 0.02

27 ± 4

no aggregation

0.46

water

6.0

219 ± 3

0.20 ± 0.01

-14 ± 1

limited aggregation

0.63

water

6.3

239 ± 2

0.19 ± 0.02

-25 ± 2

no aggregation

0.02

PBS

7.4

[0.17 ± 0.12] (*,§)

-

system unstable

0.16

PBS

7.4

-

system stable

0.25

PBS

7.4

[1666 ± 357] (*)

[0.78 ± 0.39] (*)

-

system unstable

0.46

PBS

7.4

[1032 ± 15] (*)

[0.29 ± 0.02] (*)

-

system unstable

0.63

PBS

7.4

[1027 ± 23] (*)

[0.39 ± 0.03] (*)

-

system unstable

[1797 ± 840] (*,§) 786 ± 26

0.07 ± 0.06

𝜁-potential (mV)

notes

Table 1. Characterization of CH/HA nanoparticles (NPs) after dilution 1:10 (v/v) in water and PBS. NPs were synthetized using chitosans with different fraction of acetylated units (FA). Experimental pH value of the NPs solutions, hydrodynamic diameter, aggregation at time zero, polydispersity index (PDI) and surface charge, i.e. 𝜁-potential, (all of them ±SD) of the resulting formulations are reported. (*) stands for formulations where significant signal errors in the size quality report were detected by DLS analyses; (§) stands for formulations with visible flocculated suspensions after 24 h of incubation.

As reported in Table 1, all formulations showed good homogeneity (PDI ~ 0.2), with hydrodynamic (equivalent) diameters around 200 nm for NPs prepared with chitosans with FA = 0.16, 0.46 and 0.63. At variance, NPs fabricated with chitosans with FA = 0.02 and 0.25 displayed larger dimensions. Some visible aggregates were evident only in the case of NPs synthetized with chitosan having FA = 0.46. 𝜁-potential analyses pointed at a different NPs surface charge depending on the type of chitosan: specifically, a negative surface was observed both for NPs made of chitosan with FA = 0.46 and for chitosan with FA = 0.63 (the latter being more negative); conversely, a positive surface charge was recorded for NPs composed of chitosan with FA = 0.02, 0.16 and 0.25, with values similar to what found by Parajo et al.22 Such a behavior reflects the chemical composition of chitosans, since the lower the value of FA, the higher the amount of basic glucosamines and the


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higher the (positive) polymer charge density. Therefore, a reduction of the overall basicity of chitosan upon increasing FA in turn entails an (slight but clear) increase of the pH of the corresponding salt forms upon increasing FA, as actually found (see third column of Table 1). This is confirmed by the fact that in the case of NPs prepared with chitosan having FA = 0.46, which exhibit a nearly exact stoichiometric balance of charges (see Table S1), the 𝜁-potential is already clearly negative, as a consequence of the weak basicity of chitosan. In conclusion, given the constant amount of TPP and HA in the different NPs (and the practically constant negative charge density of the latter polymer in the given pH range, i.e. independent of pH), it is of no surprise that the recorded surface net charge of NPs decrease from positive to increasingly more negative upon increasing the value of FA of the corresponding chitosan component. Another consideration can be drawn on the basis of the recent work of Goycoolea et al., where the authors attributed the reduction of the 𝜁-potential vs. FA to structural rearrangement of nanoparticles due to the higher number of hydrophobic N-acetyl-glucosamines.44 Although the procedure of preparation of NPs in water, at the slightly acidic pH value determined by the specific basicity of each chitosan (in turn depending on FA), ensured a very good dimensional homogeneity for all the investigated chitosans, still the most common condition of practical (biological) interest is that of pH = 7.4. We therefore resorted to carry out DLS experiments in phosphate buffer (PBS) at pH = 7.4. With much surprise, only NPs derived from chitosan with FA = 0.16 manifested good overall features upon dilution in PBS buffer, showing a single narrow peak in size distribution curve and a PDI value ~ 0.1 (red curve of Figure 1a and Table 1), i.e. even smaller than that at pH = 5.6 (blue curve of Figure 1a and Table 1). Actually, such low values of PDI (and high dimensional homogeneity) are difficult to reach with most of the known polysaccharide-based nanosystems, the PDI values of chitosan/hyaluronan nanoparticles being usually higher than 0.2.14,18,22,45 Higher dimensional dispersion of colloids was found for the other preparations. As a comparative sample case, the DLS curves of NPs prepared with chitosan with FA = 0.46, in water at pH = 6.0 and in PBS at pH = 7.4 are reported in Figure 1b. A significant ACS Paragon Plus Environment

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broadening and shift to large dimensions of the major dispersion curve can be noticed, together with the appearance of new distribution peaks at (very) low dimensions. A similar behavior was shown by NPs prepared with chitosan with FA = 0.63 (see Figures S1, in the Supporting Information). This behavior is suggestive of a marked heterogeneity of the system, possibly accompanied by significant swelling and aggregation, likely involving also some dissolution phenomena producing isolated polymer chains or clusters of just a few chains.

Figure 1. (a) Intensity size distribution of NPs synthetized using chitosan with FA = 0.16 and CH/HA 1:1 w/w after dilution 1:10 (v/v) at pH 5.6 in water (blue curve), at pH = 7.4 in HEPES (violet curve), at pH = 7.4 in PBS (red curve) and at pH = 7.4 in SBF (Simulated Body Fluid) (green curve), respectively. (b) Intensity size distribution of NPs synthetized using chitosan with FA = 0.46 and CH/HA 1:1 w/w after dilution 1:10 (v/v) at pH = 6.0 (in water, blue curve) and at pH = 7.4 (in PBS, red curve).

As a partial conclusion at this stage of the investigation, we can summarize that: i) in pure water, namely in salt-free conditions at the unbuffered pH value (and hence at very low total ionic strength), CH/HA NPs display a very good dimensional stability, with good value of polydispersity; ii) on the opposite, increasing pH, ionic strength (I) and phosphate concentration - up to the case of PBS - produces a general worsening of the dimensional stability. This latter feature is still somehow acceptable for the FA = 0.16 case, being the distribution curve still monomodal despite of the almost fourfold increase of the average particle hydrodynamic diameter. At variance, the new condition is highly detrimental for the cases of NPs prepared with FA = 0.02, 0.25, 0.46 and 0.63 chitosans,


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leading to the limit (or just beyond it) of the possibility of still describing those systems as reasonably homogeneous samples of spheroidal NPs, and not just as a random mixture of molecular objects, spanning from single, non-associated chains to very large, stoichiometrically undefined aggregates. All this prompted us to investigate the only case seemingly resistant to this large variation of experimental variables, namely that of NPs prepared with chitosan with FA = 0.16, in order to assess the role of each of them on NPs stability. 3.1.2 DLS characterization of NPs as a function of various physical-chemical parameters. Ionic strength, pH and phosphate concentrations are the variables that mark the observed different response of NPs on passing from unbuffered water to aqueous PBS buffer. To assess the individual contribution to such response, we started analyzing by DLS the NPs prepared using chitosan with FA = 0.16 in aqueous HEPES buffer 10 mM/NaCl 150 mM at pH = 5.6. The choice of HEPES was made to avoid the presence of even small amounts of phosphate anions, which are present in PBS and which are known to interact with chitosan. The result was a distribution curve very similar in shape to that obtained at the same pH but a very low I, with somewhat larger values of the hydrodynamic diameter and almost identical values of PDI (see Table 2).

Solvent

pH

Ionic strength (mM)

water

5.6

~1

0

207 ± 12

0.21 ± 0.01

HEPES/NaCl

5.6

150

0

321 ± 6

0.21 ± 0.01

HEPES/NaCl

7.4

154

0

384 ± 18

0.28 ± 0.05

SBF

7.4

184

1

569 ± 18

0.11 ± 0.02

PBS

7.4

168

10

786 ± 26

0.07 ± 0.06

[phosphate] (mM)

Hydrodynamic diameter (nm)

PDI

Table 2. Characterization of CH/HA nanoparticles prepared using chitosan with FA = 0.16 after dilution 1:10 (v/v) in media at different values of pH, ionic strength and phosphate concentration; hydrodynamic diameter and polydispersity index (PDI) of resulting formulations are reported.


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This result shows that the effect of the ionic strength on NPs dimensions over an almost hundredfold range, at least at pH = 5.6, is important albeit not dramatic. It stems from the screening of the attractive interactions between oppositely charged species characterizing the interpolyelectrolyte complex (polycation/polyanion) at the root of the formation of the NPs. With some surprise, however, when the DLS experiments were repeated on the same NPs using HEPES 10 mM/NaCl 150 mM, but at pH = 7.4 as medium, the dimensional distribution curve - still being nicely monomodal - shifted towards quite larger dimensions, with a similar value of PDI (see the violet curve in Figure 1a and the values in Table 2). The important role of pH was then clearly established: whereas for the polyacid hyaluronan a pH change from 5.6 to 7.4 can imply a change (increase) of the negative charge density of some percent, for the weak polybase chitosan such a change implies a probably more significant decrease of the positive charge density. As a result, the stoichiometric balance of charges in the NPs complexes passes from an excess of the positive ones at pH = 5.6 to the opposite situation of a prevalence of the negative ones at pH = 7.4 (this indirectly is supported by the trend of the 𝜁-potential values of Table 1 as function of pH, albeit for different values of FA). At present, it is not possible to quantitatively correlate the observed increase of dimensions (stemming from the prevalence of disruptive, swelling forces over the associative ones) with the decrease of charge on chitosan and its related conformational effects. Still, one can tentatively devise a mechanism to explain the observed behavior. It is conceivable that the cross-link elements holding together the NPs are of two types: the first one is traced back to the interpolyelectrolyte complex between opposing stretches of chitosan (the polycation) and of hyaluronan (the polyanion). In general, the strength of such interchain junction mainly depends on i) the charge (both as total charge and as charge density) of each opposing stretch, and ii) on the stretch extension, which directly modulated by the stiffness of each polyelectrolyte. This feature is accounted for by the total 𝑡𝑜𝑡 polymer persistence length, 𝑙𝑡𝑜𝑡 𝑝 . Two terms contribute to 𝑙𝑝 : an intrinsic persistence length, that is

determined by the intrinsic conformational features of each chain, 𝑙0𝑝 , and an electrostatic term, 𝑙𝑒𝑙 𝑝, ACS Paragon Plus Environment

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stemming from the polyelectrolyte nature of both HA and CH. 𝑙𝑒𝑙 𝑝 , depends on the polymer charge density (and then for weak polyacids and polybases on pH), and on the ionic strength of the medium. However, the interpolyelectrolyte interaction between HA and CH only is unable to give rise to well-defined spheroidal particles. It rather produces a diffuse coacervation (with tight chainchain association and massive phase separation), unless such polycation-polyanion attraction is modulated by a second cross-linking mechanism, mediated by TPP. This latter mechanism actually acts only on chitosan, being the small MW (but with high charge density and strongly acidic properties) compound TPP a very effective “point-like” chitosan cross-linker, also in the absence of any other polymer.15,46 In a sense, two competing mechanisms interplay to cross-link the polycationic CH chains: in the first one, that polyanion HA acts as the polymeric linker probably on the scale of several nanometers, in the second one the highly charged small TPP acts as a “point-like” linker, leaving a larger number of chitosan repeating units free to spatially rearrange. In unbuffered pH conditions (i.e. in slightly acidic) it is conceivable that chitosan with FA = 0.16 still bears a substantial amount of positive charge (thereby also contributing to the electrostatic stiffness via 𝑙𝑒𝑙 𝑝 ) that makes the CH chain a good partner for HA in forming associated stretches of opposite sign. This behavior of chitosan with anionic polysaccharides is known: for instance, Hsu et al. have demonstrated that “Alginate could be added into chitosan to modify the rigidity and hydrophilicity of chitosan. Higher hydrophilicity, biocompatibility, and elongation were found after modification.”47 Rising pH to 7.4 greatly weakens such cross-linking mechanism, essentially for the significant decrease of the positive charges on the chitosan chains; this latter effect is accompanied by just a slight increase of the ionization state (negative charge) of HA which at pH = 5.6 is already not far from maximum. Overall, all this greatly reduces the interpolyelectrolyte attractions; on the other side, also the “point-like” linker TPP finds a highly reduced number of -NH3+ groups on facing CH chains to be cross-linked. As a consequence, the forces stabilizing the NP structure result highly


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weakened, the osmotic swelling is less counterbalanced producing an increase of the NPs dimensions. The red curve of Figure 1a and the data of Table 1 indicate that when NPs are placed in PBS buffer at pH = 7.4, the dimensions increase with respect to the unbuffered case in water (at slightly acidic pH). In particular, for the case of particles prepared with chitosan with FA = 0.16, such increase goes well beyond the value in HEPES at the same pH (and similar I). It is straightforward to attribute such further increase to the only parameter that marks the difference between the HEPES and the PBS cases: namely, the presence of phosphate anions. Although at the given pH value the most abundant form for phosphate is the HPO42- one, i.e. definitely less charged than TPP, still the ratio phosphate:TPP is as large as 4170:1. Therefore, it seems clear that a competition exists between TPP and phosphate: as a result, the binding of the former polyanion is reduced, its crosslinking capacity is severely hampered, the osmotic swelling is even less counterbalanced and the particle dimensions increase. To verify this thesis, we resorted to perform DLS experiments in a system by all features very similar to the one based on PBS, except for having a different concentration of phosphate; simulated body fluid, SBF, is such a system, which contains phosphate at a concentration ten times lower than PBS, still at pH = 7.4. The green curve of Figure 1a and the data in Table 2 confirm that in SBF the qualitative features of the curve are the same as in HEPES and in PBS, but for the value of the hydrodynamic diameter, which falls almost exactly in between those two cases. This finding demonstrates that the phosphate anion is able to compete with the TPP one for the cationic sites of chitosan, in an extent proportional to its concentration, and to destabilize the CH/HA NPs favoring the osmotic swelling and bringing about an increase of dimensions. The dimensional shift is in line with those reported in literature for similar nano-systems. For instance, Parajo et al. reported that, after an initial increase in size, the dimensions of NPs were retained at a later time,22 albeit authors did not provide any information about the physical-chemical properties of the polysaccharides used for the synthesis of NPs to give further insights into such a ACS Paragon Plus Environment

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behavior. Comparable changes were observed for similar systems based on chitosan.13,21,48 Deng et al. reported that CH/HA nanoparticles synthetized by using polymers with similar molecular weight displayed a size increase inferior than 5% when placed in PBS buffer,49 although the size distributions, as well as the DLS signal report, were unmentioned. Additionally, the acetylation degree of chitosan was not available. As an overall comment, none of the aforementioned papers gave a clear correlation between the physical-chemical features of polysaccharides and properties (e.g. PDI) of the resulting nanoparticles when dispersed in media with approximately neutral pH and physiological osmolarity. Moreover, the potential role of the phosphate anion apparently has not been given the necessary attention. Herein we want also to stress that the variation of the chitosan acetylation degree could represent a powerful tool for regulating NPs stability. In conclusion, the chitosan sample with FA = 0.16 seems to be the best candidate for the preparation of NPs with respect to samples with higher or lower level of acetylation as to the dimensional stability considering the final fate of NPs in physiological conditions.

3.2. Biological characterization 3.2.1 Effect of NPs on macrophage viability Neutral Red assay was performed to assess the cytotoxicity of NPs toward cultured human macrophages. Statistical analyses did not show significant differences between the viability of cells treated with NPs and control, thus pointing up the good biocompatibility of NP preparations (Figure S2). The lack of toxicity of these NPs is in line with data previously published by other authors which used polymers with similar molecular properties (i.e. degree of acetylation and/or molecular weight).12,22,45,50 Furthermore, none of the NP components tested separately (i.e. CH and HA/TPP) displayed cytotoxic effects (data not shown), at variance with some literature data reporting that some chitosans would cause cellular damage.51 Moreover, although it has been reported that high molecular weight HAs should stimulate cell proliferation,52 this was not the case in our system, given the comparatively quite lower molecular weight used for the synthesis of NPs. ACS Paragon Plus Environment

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3.2.2 Effect of NPs on TNF-α production by macrophages To study the influence of NPs and of their components on the production of pro-inflammatory cytokines by human macrophages, the release of TNF- in the supernatants of differentiated U937 cells was assessed after 3 h of treatment. As shown in Figure 2, macrophages displayed a very low basal level of TNF- production (black column) that significantly increased in cells treated with a concentration 1 ng/mL of LPS (grey column). HA/TPP and chitosans with FA = 0.02 and 0.25 mildly elicited TNF- production, whereas other chitosans with different FA did not significantly modify the basal TNF- production (Figure S3). LAL test on FA = 0.25 chitosan sample evidenced an endotoxin level of 309 EU/g which could explain the slight increase in TNF- release. On the opposite, LAL analysis on FA = 0.02 chitosan showed a residual endotoxin level of only 24 EU/g, thus suggesting that a physical effect - i.e. the remarkable insolubility of this type of chitosan at physiological pH and its ensuing precipitation - rather than the minimal endotoxin contamination would lightly stimulate macrophages. When the cells were treated with NPs, only those made of FA = 0.16 chitosan did not stimulate TNF- production both in basal (Figure 2) and in LPS-stimulated conditions (Figure S4). Conversely, NPs synthetized with other chitosan samples significantly stimulated the basal production of TNF-α.


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Figure 2. TNF- production by U937 macrophages treated with the indicated substances (final concentrations were 80 μg/mL for NPs and 1 ng/mL for LPS) for 3 h. Results were normalized for the number of viable cells. Data are means (±SD) of three to ten measurements. Student’s t-test: NS, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0005.

By considering the above reported stability studies (see paragraph 3.1.1), it can be hypothesized that the partial or severe instability of NPs prepared using chitosans with FA = 0.02, 0.25, 0.46 and 0.63 with ensuing loss of structural components stimulate the production of TNF-α. This evidence is clearly supported by the slight pro-inflammatory activity of singular HA/TPP mixture plus some chitosan samples and by the dimensional heterogeneity of unstable NPs (see DLS discussion). In the case of NPs constituted of FA = 0.46 chitosan, this condition results exacerbated: the TNF-α production was in fact as prominent as that of positive control LPS, due to the fully instability of resulting colloids upon dilution in physiological-mimicked media (Figure 1b) and consequent release of the HA/TPP mixture. However, the main contribution to such a behavior seems being originated from the presence of truly heterogeneous aggregates/precipitates - already visible to the naked eye in pure water (see Table 1) - and residual entangled polysaccharide chains, which jointly elicit a pro-inflammatory response. The finding that NPs made of FA = 0.16 chitosan did not alter the basal TNF- production, is in contrast with the data reported by Almalik et al., where a TNF- production of about 10 times as high as that of untreated cells was noticed using similar NPs (obtained by using chitosan with FA = 0.15),45 that could be likely ascribed to the


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different cellular model used. Given the excellent performance of NPs prepared with FA = 0.16 chitosan in terms of both macrophage viability and absence of undesired increase in basal TNF-α secretion, at variance with particles prepared with chitosan of diverse FA values, all the subsequent experiments were carried out with the aforementioned chitosan.

3.2.3 Membrane association and internalization of NPs by macrophages The membrane-association to and cellular uptake by U937 cells of fluorescence-labeled NPs were investigated by means of flow-cytometry (Figure 3). Immediately after the addition to the cell suspension of NPs prepared using chitosan with FA = 0.16, a significant shift of the green fluorescence signal was evident (Figure 3b), thus indicating that the vast majority of cells (96.8%) interacted with the particles. This interaction was very fast, similar to that already reported to occur between neutrophils and similar NPs.29 This rapid interaction could be accounted for by receptormediated association (e.g. CD44) for both the polysaccharides,18,19 but also ascribed to (weak) electrostatic interactions between positively charged NPs and negatively charged cells. The internalization of NPs was very low up to 3 h of incubation (Figure 3d), since the green fluorescent cell subsets in the upper plus lower right quadrants were less than 1%. Furthermore, at this incubation time, NPs-cell association was weak, since low speed centrifugation included in the procedure of recovering the cells from the wells was sufficient to cause complete NPs detachment, as occurred in the case of human neutrophils.29 Similar results were obtained mimicking a proinflammatory condition, i.e. using LPS-stimulated cells and a medium supplemented with nondecomplemented serum (data not shown).


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Figure 3. Membrane association and internalization of fluoresceinamine isomer I-labeled NPs (chitosan with FA = 0.16 and CH/HA 1:1 w/w) by U937 macrophages. (Panels a, b): Green fluorescence (FL-1) for control cells (a) and for NPs-treated cells immediately before the cytometric analysis (b). (Panels c, d, e): Dot plot of green (FL-1) and red (i.e., TB-quenched) fluorescence (FL-3) of control cells (c), cells incubated with NPs for 3 h (d) and for 24 h (e). Four cell subsets are identifiable: lower left quadrant, no interaction; upper left quadrant, association; upper right quadrant, association and internalization; lower right quadrant, internalization. The number of events falling in each quadrant is expressed as a percentage of total gated events. Macrophages were incubated at 37 °C with NPs (80 μg/mL final concentration) or not (control). Before being analyzed, samples were diluted with a TB (Trypan Blue) solution to quench the green fluorescence. Samples were washed after the incubation with NPs.

After 24 h of incubation the cell subset bearing internalized NPs drastically increased (Figure 3e): nearly 60% of cells were positive to fluorescence emitted by engulfed NPs, thereby indicating that particle internalization is a slow-paced event.50 The slow internalization process of NPs by macrophages could be seen as an advantage for the delivery of molecular therapeutics that must be released in the extracellular space and exert their activity from the outside of their cellular targets. In fact, a too rapid sequestration of the carrier would compromise the efficacy of the drug treatment.

3.2.4 Effect of NPs on neutrophil H2O2 production, adhesion to FBG and granule release The finding that NPs made with FA = 0.16 chitosan did not stimulate TNF- production by


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cultured human macrophages, prompted us to run experiments aimed at assaying the effect of these particles on the functional response of neutrophils, another type of professional phagocytes. In particular, neutrophil adherence to biologic surfaces and ROS production were analyzed at first. Adherence to endothelial cells and extracellular matrix components has been shown to play a crucial role not only in neutrophil recruitment to the inflamed microenvironment but also in the regulation of their functional responses, among which TNF-induced ROS production is one of the most widely studied.53 Figure 4 shows that, after 1 h of incubation with neutrophils, NPs affected neither adhesion to FBG-coated surfaces nor H2O2 production (Figure 4a and 4b, respectively). Moreover, NPs did not modify the amount of MPO released from the cells (Figure 4c). Collectively these findings thus suggest that such particles do not interfere with the process of neutrophil recruitment to the inflamed sites and, in the meantime, they do not act, per se, as a stimulus for an enhanced ROS production and degranulation, two major neutrophil functional responses, which contribute to inflammation-induced tissue damage.


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Figure 4. Effect of NPs (chitosan with FA = 0.16 and CH/HA 1:1 w/w) on neutrophil adhesion (%) (a), H2O2 production (b) and myeloperoxidase (MPO) release (c). (a,b): neutrophils were incubated for 60 min with NPs at 37 °C in FBG-coated wells. In (b) results are expressed as HRP-mediated, H2O2-dependent DHR oxidation. Data have been normalized for the number of adherent cells. Data are means (±SD) of six measurements. Student’s t-test: NS, not significant. (c) Neutrophils were incubated with NPs for 60 or 90 min at 37 °C in FBG-coated wells. On completion of the incubation, the plates were centrifuged and aliquots of the supernatants were assayed for MPO activity as detailed in Materials and Methods section. The grey column shows the amount of MPO released by neutrophils stimulated with 5 x 10-7 M fMLP, a known neutrophil secretagogue. Data are means (±SD) of four measurements. Student’s t-test: NS, not significant.

3.3 Evaluation of muco-adhesive properties To explore the muco-adhesive properties of NPs, an analysis of their interaction with mucin was undertaken. Specifically, variation of particle size distribution was monitored by means of DLS measurements.43 When NPs and mucin were mixed, a progressive increase of NPs size distribution


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towards larger dimensions was noticed, proportional to the amount of mucin (Figure S5), indicating that the glycoprotein interacted with NPs in a dose-dependent manner. In parallel, PDI values gradually increased (Figure S5a), suggesting the formation of more heterogeneous aggregates. Similar results were obtained also at pH = 7.4 (data not shown). The nature of such interactions could be traced back to the electrostatic forces arising between the positive surface charge of NPs and the negatively charged mucin.54 Hydrogen bonding and hydrophobic effects could also be considered important contributions for modulating NPs muco-adhesiveness. Although more sound conclusions on NPs muco-adhesiveness could be drawn only from more extensive experiments carried out using more complex setups, the simple evidence that mucin interacts with the NPs is a strong clue in favor of the possibility of attributing them such a feature.

4. Conclusions In this work, the role played by the acetylation degree of chitosans for the synthesis, stability and performance of NPs in association with hyaluronan and TPP was studied. Specifically, chitosans with different fraction of acetylated units, FA, namely 0.02, 0.16, 0.25, 0.46 and 0.63, fostered the formation of particles in the nano-size range with good homogeneity. When placed in physiological-simulated media in terms of pH and osmolarity, such as PBS or SBF, only NPs made with FA = 0.16 chitosan maintained their integrity without dissolution or severe aggregation. Resulting NPs were analyzed in terms of activation of immune system cells, namely macrophages and neutrophils. More in detail, NPs composed of FA = 0.16 chitosan had not effect on TNF- production by macrophages whereas the other formulations enhanced such a production. In line with these results, NPs made with FA = 0.16 chitosan did not modify neutrophil adhesion to solid substrates, H2O2 production and MPO release. Flow cytometry studies proved that most of the macrophages interacted with NPs immediately after their addition to the culture medium, albeit this association was weak. Furthermore, only negligible fraction of cell population internalized NPs after 3 h of incubation, whereas most cells displayed engulfed particles after 24 h.


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Recapitulating, our studies undoubtedly prove that (i) chitosan acetylation degree dictates NPs dissolution/aggregation stability, (ii) selected CH/HA NPs are a stable and homogeneous ensemble of colloids, (iii) show lack of cytotoxicity, (iv) do not elicit any pro-inflammatory response and, (v) are barely engulfed in the early stages of incubation with macrophages. Hence, they represent promising carriers for drug delivery of molecule therapeutics to treat pathological conditions, e.g. chronic inflammatory diseases. With regard to this point, the muco-adhesive property could provide further benefits to these NPs, especially in the treatment of inflamed mucous membranes of respiratory, gastrointestinal and urinary tracts.

Supporting information The Supporting Information is available free of charge on the ACS Publications website. (i) Molecular features of chemicals used for the synthesis of nanoparticles (NPs); (ii) Physical/chemical characteristics of chitosans used in this study; (iii) Intensity size distribution of NPs synthetized using chitosan with FA = 0.63; (iv) Neutral Red assay on U937; (v) TNF- production by LPS-stimulated macrophages; (vi) Interaction between NPs and mucin.

Notes The authors disclose any actual or potential conflict of interest.

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

15

stable NPs

10 5 0 10

8


100

unstable NPs

chitosan

intensity (%)

intensity (%)

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6 4 2 0 10

1000

100

1000

size (nm)

size (nm)

TNF-‐α no eﬀect


pro-‐inﬂammatory s9mulus

Chitosan acetylation degree influences the physical properties of

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