Sandra Robla

Maruthi Prasanna

Rubén Varela-Calviño

Cyrille Grandjean

Noemi Csaba

Rubén Varela Calviño

A chitosan-based nanosystem as pneumococcal vaccine delivery platform

Keywords: Chitosan, nanovaccine, dendritic cell, antigen protein, immune response

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the human upper respiratory tract and causes pneumonia, meningitis and septicemia [START_REF] Pérez-Dorado | Pneumococcal surface proteins: When the whole is greater than the sum of its parts[END_REF]. It is responsible for high morbidity and mortality rates, especially among infants, elderly and immunocompromised people [START_REF] Singh | Prediction and characterization of helper T-cell epitopes from pneumococcal surface adhesin A[END_REF]. The cell surface of S. pneumoniae contains various proteins and capsular polysaccharides (CPS) that play an important role in pathogen attachment and immune system evasion. The CPS diversity results in more than 96 stereotypes whereas cell surface proteins are more conserved and less variable [START_REF] Mohammadzadeh | Recent approaches in whole cell pneumococcal vaccine development : a review study[END_REF]. Currently available CPS-based vaccines such as PCV13 and PCV10 (Pneumococcal Conjugated Vaccines) and PPSV23 (Pneumococcal Polysaccharide Vaccine) only induce specific serotype dependent immune responses [START_REF] Leites | Cloning and optimization of induction conditions for mature PsaA ( pneumococcal surface adhesin A ) expression in Escherichia coli and recombinant protein stability during long-term storage[END_REF]. In addition, PPSV23 lacks the ability to generate robust immune responses in high-risk populations such as infants or the elderly. To address some of these obstacles, several studies investigated the possibility of using protein antigens expressed at the S. pneumoniae cell wall as potential vaccine candidates. One relevant cell membrane protein is Pneumococcal surface adhesin A (PsaA) [START_REF] Bartual | Three-dimensional structures of Lipoproteins from Streptococcus pneumoniae and Staphylococcus aureus[END_REF]. PsaA is a critical factor for the colonization and adhesion of the pneumococcus in nasopharyngeal epithelial cells. It has been shown to activate both the humoral and cellular response of the immune system against the entry of the pathogen via the respiratory airways [START_REF] Rajam | Pneumococcal surface adhesin A (PsaA): A review[END_REF].

Intranasal route has been extensively explored for non-invasive immunization, stimulating mucosal and systemic immunity and protecting against respiratory infections [START_REF] Zheng | Noninvasive vaccination against infectious diseases[END_REF]. The protective nasal epithelial barrier presents a large surface area which produces a rapid permeation of drugs [START_REF] Almeida | Chapter 3.1. Nanocarriers Overcoming the Nasal Barriers: Physiological Considerations and Mechanistic Issues[END_REF], as well as the NALT (Nasal Associated Lymphoid Tissue), responsible for the production of long-term antigen-specific antibodies [START_REF] Calvo | Chitosan and chitosan/ethylene oxide-propylene oxide block copolymer nanoparticles as novel carriers for proteins and vaccines[END_REF][10]. The nasal barrier exhibits metabolic activity and is covered with a dynamic mucus layer which limits the permeability of the epithelium and decreases formulation residence time [START_REF] Illum | Nasal drug delivery -Possibilities, problems and solutions[END_REF]. In this context, nanostructures have been described as interesting tools to enhance the delivery of proteins, improving the immunogenicity of nasally administered vaccines [START_REF] Slütter | Rational design of nasal vaccines[END_REF]. Indeed, nanostructures play an important role in the stimulation of an adaptive immune response, upon facilitating the antigen uptake and processing by professional antigen-presenting cells such as dendritic cells (DCs) [START_REF] Lewis | Dendritic cells: Arbiters of immunity and immunological tolerance[END_REF], and the immediate innate immune response against these inhaled external substances by macrophages [START_REF] Cole | The Role of Macrophages in the Innate Immune Response to Streptococcus pneumoniae and Staphylococcus aureus[END_REF]. Chitosan (CS), a polysaccharide derived from the deacetylation of chitin [START_REF] Prego | The potential of chitosan for the oral administration of peptides[END_REF] has been widely used in the design of nanocarriers for nasal absorption of peptides like insulin, salmon calcitonin, leuprolide or parathyroid hormone [START_REF] Prego | Chitosan nanocapsules: A new carrier for nasal peptide delivery[END_REF], thanks to its high mucoadhesiveness, biocompatibility and biodegradability [START_REF] Kean | Biodegradation, biodistribution and toxicity of chitosan[END_REF]. Previous studies have shown that chitosan nanocapsules are good candidates for protein administration by nasal route [START_REF] Prego | Chitosan nanocapsules: A new carrier for nasal peptide delivery[END_REF], and also have been reported to have an adjuvant effect by promoting innate immune responses [START_REF] Shi | Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine[END_REF].In this research, a mucoadhesive chitosan nanocarrier containing a covalently conjugated pneumococcal protein antigen was designed and characterized to evaluate its capacity as a nasal drug delivery system..

Materials and methods

Miglyol 812® was purchased from Cremer Oleo (Hamburg, Germany), polyethylene glycol stearate 40 (PEG-st 40) was purchased from Croda Chemicals Europe Ltd (Snaith, United Kingdom) and sodium glycocholate (SGC) was obtained from Dextra laboratories Ltd (Reading, United Kingdom). Chitosan HCl ((C6H11NO4Cl) n, Mw: 1526.5 g/mol, deacetylation degree of 85%) was purchased from Heppe Medical Chitosan GmbH (Saale, Germany). provided by BioVision (CA, USA). Triton ® X-100 was purchased from Scharlab (Barcelona, Spain), and interferon gamma (IFNɣ) was purchased from Peprotech, (London, UK). All other chemicals were of reagent grade or higher purity.

Antigen production and characterization

The mature PsaA used as an antigen was previously expressed and purified [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF]. Briefly, a plasmid encoding PsaA deprived of its signal peptide was transformed into E. coli BL 21 cells. Fractions containing the target protein were purified by affinity chromatography and the protein was purified to eliminate any traces of endotoxin present using endotoxin removal beads.

Functionalization of the polymer and proteins and their characterization

PsaA-SATA conjugation

For functionalization of PsaA, 1.35 mg of SATA was incorporated at 45 minute-intervals for 3 times (0.45 mg x 3, ~40 equiv) into 5 mg/mL of PsaA antigen in PBS 0.1 M pH 7.4, at 4ºC under stirring. Subsequently, the reaction mixture was dialyzed in 0.1 M PBS to eliminate the excess of reagent using dialysis membrane (3.5 kDa) at 4°C. The ability of the conjugated protein to retain its structural conformation was studied using circular dichroism (CD) using Jasco J-810 spectropolarimeter. The baseline was obtained using a blank solution containing the buffer. Solution of PsaA or the conjugated PsaA-SATA was diluted to 0.1 mg/mL in buffer solution and was scanned over the wavelength range 200-260 nm. The analysis was performed using a 1-nm quartz cylindrical cell at a scanning speed of 50 nm/min, 0.1 nm bandwidth and a temperature of 20°C. PsaA and PsaA-SATA mass spectra were also determined on an Autoflex III MALDI-TOF/TOF spectrometer (Bruker Daltonics).

Chitosan-maleimide conjugation

In brief, maleimide-propionic acid (0.553 mg, 5:1 ratio CS:m-PA) in dimethyl sulfoxide (DMSO) was reacted with 5 mg of chitosan in the presence of 0.339 mg of NHS (2:1 ratio CS:NHS) and 0.188 mg of EDC (2:1 ratio CS:EDC) as crosslinkers and were kept under constant stirring for 24 h. Excess reagents were removed by dialysis using dialysis membrane (3.5 kDa). The chemical conjunction between chitosan and maleimide-propionic acid was confirmed by H 1 -NMR spectrometry (Bruker BRX-500).

Preparation of the nanocapsules

Blank CS and CS-maleimide nanocapsules (NCs) were prepared by solvent displacement technique [START_REF] Grenha | Chitosan nanoparticles: A survey of preparation methods[END_REF].

An organic phase of 1.6 mg of PEG st-40, 0.5 mg of SGC and 6.25 µL of Miglyol ® (density 0.94 g/mol) in a final volume of 500 µL of ethanol was poured over a 0.5 mg/mL CS (for CS NCs obtention) or CSmaleimide (for CS-maleimide NCs) solution [START_REF] González-Aramundiz | Rational design of protamine nanocapsules as antigen delivery carriers[END_REF], and was kept under continuous stirring for 1 h. Later, the organic solvent was removed under vacuum using rotary evaporation (R-300 Buchi ® , 150 rpm, 50 mbar, 5 min at 37°C) to a constant volume of 1 mL. Finally, nanocapsules were isolated by centrifugation (Eppendorf 5430R ® , at 20.000 g for 30 min at 4°C) and resuspended in ultrapure water.

The association of PsaA and PsaA-SATA was carried out at the end of the synthesis of CS and CSmaleimide nanocapsules, respectively, using a mass ratio of 1:5 (PsaA:polysaccharide) for the association of the protein. In both cases, protein was added and kept under continuous stirring for 10 min, and to ensure the entire removal of unassociated PsaA/PsaA-SATA, nanocapsules were centrifugated (10.000 g for 5 min at RT).

For in vitro studies, the conjugated NCs were labeled with the fluorescent marker Cy5. For this, Cy5-NHS in DMSO was added to CS-maleimide in acetate buffer at pH 5.6 (mass ratio 1:1) and kept overnight under magnetic stirring. Then, the reaction was stopped, and excess dye was removed using PD10 columns (Centri-Pure P10-Gel Fixing Column, Zetadex-emp Biotech GmbH, Berlin, Germany) and the purified product was dialyzed again overnight.

Optionally, NCs were freeze dried in the presence of trehalose 5% (w/v) as cryoprotectant. NCs and trehalose solution were mixed 1:1 (v/v) in a freeze-drying glass vials, they were frozen at -80°C and then transferred to the freeze-drier (Labconco Corp., USA), using a primary drying cycle from -40ºC to 20ºC (+10ºC/step, 18h, 12h, 5h and 4x2h) and a secondary drying cycle (22ºC, 3 h and -20 mTorr vacuum conditions).

Physicochemical characterization of nanocapsules

The hydrodynamic diameter, polydispersity index (PDI) and derived count rate of the prepared NCs were measured using dynamic light scattering (DLS). The zeta potential values were obtained by laser droppler anemometry using a Zetasizer Nano-S (Malvern Instruments; Malvern, UK). The NCs were diluted 1:20 in ultrapure water (MilliQ ® , Merck, NJ, USA) prior to the measurement. Analysis was performed in triplicate at 25°C with a detection angle of 173° in ultrapure water. Nanoparticle tracking analysis (NTA) was used to measure the particle size of NCs based on imaging after dilution in ultrapure water using a NanoSight NS3000 equipment (Malvern analytical Ltd., Malvern, UK). For NTA analysis CS NCs were diluted 1:100 to keep the concentration within the instrument measuring range.

Association efficiency

The association efficiency (AE%) of protein was estimated by quantifying the difference in absorbance of the total amount of protein and the amount of free protein present in the supernatant after centrifugation.

The free protein in the supernatant was analyzed by Micro BCA assay colorimetric method (Micro BCA Protein Assay, Thermofisher Scientific, Spain) using a microplate reader (Synergy H1 Hybrid Multi-Mode, BioTek, Winooski, VT) by measuring the absorbance at 562 nm.

Morphological characterization

The surface morphology of the nanocapsules was studied using FESEM and STEM electron microscopy (FESEM Ultra Plus, ZEISS, Germany). For FESEM studies, 10 µL of the diluted sample was placed on the silicon wafer and kept in the desiccator overnight for drying. Before the analysis, the samples were sputter coated with iridium in an argon atmosphere. For STEM studies, NCs were also diluted 1:100 and 10 µL was placed on copper grids with carbon films and then washed drop-by-drop with water. Later the grids were air dried overnight in the desiccator and the analysis was performed.

Colloidal stability

The colloidal stability of the antigen loaded nanocapsules before and after freeze drying procedure was studied in vitro using Simulated Nasal Fluid (SNF), composed of 7.45 mg/ml of NaCl, 1.29 mg/ml of KCl and 0.32 mg/ml of CaCl2•2H2O and pH adjusted at 6.5. SNF supplemented with mucin type III (1% sialic acid content) at 0.1% was also used for the study. For further studies in dendritic cell culture, the stability of the nanosystems was assessed in cell culture RPMI-1640 medium, following the change in particle size, mean count rate and PDI over a period of 24 h. In brief, 20 μl of NCs were mixed with 980 µL of SNF with 0.1% mucin or 980 μl of RPMI, while nanoparticles suspended in SNF and water at same concentrations are used as controls. The NCs in the different media were shaken at 100 rpm at 37°C using a constanttemperature shaker, at different time points (0, 6 and 24 h) the size and count rate of samples were measured using DLS as described in Section 2.3.1.

Release studies

In vitro release studies of PsaA from the conjugated nanocapsules was performed by incubating nonconjugated CS-PsaA and conjugated CS-maleimide PsaA nanocapsules in a final volume of 1 mL of SNF in a shaking incubator (Biosan Termo-Shaker TS-100C, 600 rpm at 37°C). At different time intervals (1, 3, 5, 8, 12 and 24 h), samples were centrifuged (5 min at 10,000 g and RT) and 250 µL of supernatant were collected for analysis and replaced with fresh SNF medium to maintain sink conditions [START_REF] Shen | In vitro dissolution testing strategies for nanoparticulate drug delivery systems: recent developments and challenges[END_REF]. The amount PsaA contained in the supernatant at scheduled time intervals were subsequently analyzed by absorbance measurement in a multilabel plate reader using BCA assay at 562 nm.

Blood samples and dendritic cell preparation

Heparinized blood samples were obtained from healthy donors after informed consent. The buffy coats from anonymous donors were donated by the Organ and Blood Donation Agency (ADOS; Santiago de Compostela. Spain). The peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll gradient centrifugation method [START_REF] Posch | Generation of human monocyte-derived dendritic cells from whole blood[END_REF]. Briefly, blood was diluted with PBS (1:1) and layered onto Ficoll-Paque™ PLUS at a ratio of 2:1 (blood:Ficoll). Different components were isolated after centrifugation (Allegra X-12R, Beckman Coulter) at 400 g for 30 min at RT, and PBMCs were collected from the interface, washed twice with PBS and centrifuged again (300 g, 10 min at RT). Cells were resuspended in R2 medium (RPMI-1640 supplemented with 2% heat-inactivated FBS and 1% of PSG). The density was adjusted to 1×10 6 cells/mL and 10 mL of cells were seeded into 75 cm 2 cell culture flask for 2 h (37°C, 5% CO2). After the incubation period, non-adherent cells (peripheral blood lymphocytes or PBLs) were washed with PBS and attached monocytes were cultured for another 6 days, renewing half of the medium after three days, in R10 medium (RPMI-1640 supplemented with 1% antibiotics and 10% FBS) in the presence of IL-4 and GM-CSF (both at 100 ng/mL) to allow the cells to differentiate into DCs [START_REF] Banchereau | Immunobiology of Dendritic Cells[END_REF][25].

NCs toxicity on immune cells

MTS assay

The cytocompatibility of the antigen loaded CS-maleimide PsaA NCs with the dendritic cells was studied using MTS assay, and the presence of the soluble formazan product was determined by measuring the absorbance that is directly proportional to the number of viable cells [START_REF] Hu | The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide: Pidotimod(PTD)[END_REF]. Monocyte differentiated immature DCs (iDCs) were added into 48-well microplate (1x10 6 cells/mL) and blank CS-maleimide and CS-maleimide PsaA NCs with a concentration range from 400 μg/ml to 25 μg/ml were incorporated. As a negative control (0% toxicity), iDCs in culture medium was used, whereas, for the positive control (100% toxicity), iDCs were incubated with Triton X-100 (0.5%). In the same manner, macrophage Raw 264.7 cells were incubated with blank CS-maleimide and CS-maleimide PsaA NCs and treated using the same procedure as iDCs. After 4 and 24 h of incubation under standard sterile conditions for cell culture (37°C and 5% CO2), cells were collected and centrifuged (Microfuge 5427-R, Eppendorf, 500 g, 5 min at RT) for the removal of un interacted nanocapsules. Finally, the cells were incubated with 10 µL of MTS reagent and the absorbance was measured at 490 nm using a microplate reader.

7-AAD assay

Cytotoxicity studies on the dendritic cells were also carried out using the intercalative DNA fluorescent dye 7-AAD. iDCs were incubated for 24 h with different CS-maleimide PsaA NCs concentrations from 900 µg/mL to 50 µg/mL and after the incubation period, cells were washed and stained with 0.05 μg/μL of 7-ADD (30 min at 4°C) for analysis by flow cytometry (BD FACSCalibur™, Becton Dickinson). The emission of 7-AAD was detected in the FL-3 channel at 650 nm. The analysis was performed with Flowing software (Cell Imaging Core, Turku Biotechnology Center, Turku, Finland) [START_REF] Lecoeur | Multiparametric flow cytometric analysis of biochemical and functional events associated with apoptosis and oncosis using the 7-aminoactinomycin D assay[END_REF].

Interaction of NCs with immune cells

The analysis of the uptake of CS-maleimide PsaA NCs by the DCs and Raw 264.7 cells was processed by means of confocal laser scanning microscopy (CLSM) and FESEM microscopy, respectively. For CLSM, DCs were incubated with Cy5-labeled nanosystems at a concentration of 150 μg/ml and they were visualized by confocal microscopy (Leica SP5, Mannheim, Germany) after 24 h. For FESEM analysis, Raw 264.7 cells were seeded on a poly-L-Lysine coated cellware (Corning Life Sciences, MA, USA) and nanocapsules were added, and allowed to incubate for 24 h. Later, the cells were fixed using 4% paraformaldehyde (PFA), washed and gradually dehydrated using increasing ethanol concentrations (30%, 70%, 96%, 100%). Cells were placed in the desiccator overnight and were sputter coated with iridium before visualization [START_REF] Hu | The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide: Pidotimod(PTD)[END_REF].

Human DCs phenotype analysis

iDCs were incubated with the conjugated CS-maleimide PsaA NCs at a final concentration of 150 μg/ml during 48 h. Cells were washed twice (400g, 6 min at RT) to eliminate excess NCs, resuspended in PBS and stained with optimal amounts of different antibodies (CD1a-FITC, CD83-APC and CD86-PE) for 25 min at RT, in the dark. Finally, levels of maturation markers were quantified by flow cytometry.

2.9. NCs tolerogenic effect in immature DCs 2,3-Indoleamine dioxygenase (IDO) expression was evaluated in iDCs maturation after conjugated CSmaleimide PsaA NCs exposition. In brief, 1.25 μl of L-tryptophan (100 μM) was added to the iDCs medium during the last 4 h of incubation. Then, cells were centrifuged (1,000 g, 5 min at RT) and supernatants were mixed with 30% trifluoroacetic acid (2:1 v/v) and centrifuged again (10,000 g, 5 min, RT). The supernatant was mixed with the Ehrlich Reagent (1:1 v/v) and absorbance at 490 nm was determined using a microplate reader as indicated.

Activation of CD4 and CD8 T cells by NCs

After total monocyte differentiation into iDCs, they were harvested, resuspended in R10 medium and iDCs (1x10 6 cells/mL) were co-incubated with 150 μg/ml of blank CS-maleimide NCs, CS-maleimide PsaA NCs and PsaA. As a negative control (no activation), iDCs in culture medium were used, whereas for the positive control (total activation), mature DCs (mDCs) were obtained by incubation of iDCs with bacterial LPS (20 ng/ml) and IFN-γ (200 IU/ml). After 48 h, cells were harvested and centrifuged to remove nanocapsules (300 g, 10 min at RT). In parallel, allogenic PBLs were obtained from buffy coats after 2 h of PBMCs incubation with R2 medium and they were co-incubated with DCs for 12 days, renewing culture media and splitting the cells as necessary (usually every 2-3 days). Non-adherent cells were collected and centrifuged (500 g for 5 min at RT), washed and divided into two different groups. CD4-APCs together with CD25-PE (5 µL/each) were added to the first, while CD8-APCs together with CD28-PE (5 µL/each) were added to the latter and left to incubate at 4°C for 25 min in darkness. After incubation, cells were washed and centrifuged (400 g for 6 min at RT) for their analysis by flow cytometry as described above.

2.11.

Cytokine secretion by PBMCs incubated with different NCs

PBMCs (1x10 6 cells/mL) were collected from healthy donors and incubated with blank CS-maleimide NCs, CS-maleimide PsaA NCs and PsaA alone (150 µg/mL) (5% CO2, 37 °C) for 2 h. After that, cells were washed and centrifuged (300 g for 10 min at RT), thus removing the nanocapsules. Then, BFA (10 mg/ml, 5 µL/well) was added to block the secretion of cytokines and kept for overnight incubation. The following day, cells were centrifuged again under the same conditions, the PBMCs surface was stained with CD3-FITC (10 µL/well) antibody and left for 15 min at 4°C in darkness. Cells were washed, centrifuged (400 g, 6 min at RT) and fixed with 1% PFA under constant agitation to avoid cell aggregation, and were incubated again for 15 min in darkness. Cell membrane was permeabilized with 1% saponin and centrifuged again under the initial conditions for an intercellular staining with TNFα-PE cytokine antibody (5 µL/well) and incubated for 15 min at 4°C in darkness. Finally, cells were washed twice with 0.1% saponin buffer and fluorescence signals were measured by flow cytometry as indicated.

Statistical analysis

Results were expressed as mean ± standard deviation (SD) and mean ± standard error mean (SEM) for dendritic cells studies. Data analyses were carried out using GraphPad Prism v.6.01 software (GraphPad Software Inc., San Diego, CA, USA). Statistical analyses using the 2-way ANOVA test in combination with Dunnett's multiple comparisons test were used to determine the difference between donor groups.

Results and discussion

3.1. Functionalization of the polymer and protein and their characterization

3.1.1. PsaA-SATA conjugation
In the search for a serotype-independent pneumococcal vaccine, the use of highly conserved pneumococcal protein antigens has become an attractive alternative to achieve immune response against S. pneumoniae [START_REF] Moffitt | Rationale and prospects for novel pneumococcal vaccines[END_REF]. PsaA, a 35-kDa lipoprotein present in all serotypes known to date, is capable of inducing herd immunity against the pathogen [START_REF] Tai | Streptococcus pneumoniae protein vaccine candidates: Properties, activities and animal studies[END_REF], and studies of its co-administration with PCV7 have demonstrated to increase serotype coverage and immunogenicity of PCV7 by reducing nasopharyngeal carriage [START_REF] Whaley | Concomitant administration of recombinant PsaA and PCV7 reduces Streptococcus pneumoniae serotype 19A colonization in a murine model[END_REF].

In our study, the PsaA was obtained by rDNA technology and subsequently, the free amines present in the lysine amino acids of the PsaA were derivatized to produce a thiolated protein. The functionalization with SATA adds sulfhydryl groups to proteins in a protected form. Thus, PsaA when modified with SATA can be stored for extended time periods and the sulfhydryl groups required for the conjugation reactions will be exposed by treating with the hydroxylamine. Conjugation reaction between the PsaA protein and the SATA group is described in Supplementary Figure 1a. The MALDI-TOF technique (Figure 1a) used to determine the protein molecular weight showed that the purified PsaA had a mass of 34,059 Da, while

PsaA-SATA conjugated protein had a mass of 35,683 Da. The difference in the molecular weight between

PsaA and PsaA-SATA confirms the successful conjugation of SATA to PsaA antigen in the ratio of 13:1.

The presence of multiple sulfhydryl groups on the protein helps its efficient coupling to the nanocapsules.

Similarly, Derksen et al., have described the use of protein functionalized with SATA for its surface conjugation to liposomes containing maleimide residues [START_REF] Derksen | An improved method for the covalent coupling of proteins to liposomes[END_REF]. In addition to this, the sulfhydryl modified proteins were also used to prepare protein-protein conjugate nanoparticles to enhance the immunogenicity of the protein antigens [START_REF] Scaria | Protein-protein conjugate nanoparticles for malaria antigen delivery and enhanced immunogenicity[END_REF]. The conformation of the protein antigen is pivotal in conserving the potential epitopes that are responsible for the immunogenicity [START_REF] Singh | Prediction and characterization of helper T-cell epitopes from pneumococcal surface adhesin A[END_REF]. The CD spectra (Figure 1b) revealed no changes in the secondary structural conformation of the PsaA-SATA compared to the existing literature [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF]. The PsaA antigen presented negative peaks at 208 and 221 nm, which were also observed in the conjugated PsaA-SATA antigen, indicating a similar protein folding pattern of alpha helixes and beta sheets in both cases [START_REF] Prasanna | Semisynthetic glycoconjugate based on dual role protein/PsaA as a pneumococcal vaccine[END_REF][START_REF] Couñago | Imperfect coordination chemistry facilitates metal ion release in the Psa permease[END_REF]. PsaA-SATA conjugate.

Chitosan-maleimide conjugation

Chitosan is an attractive biomaterial with amine groups for conjugation of ligands [START_REF] Sahoo | Chitosan: A new versatile bio-polymer for various applications[END_REF], and previous studies have reported its successful covalent conjugation with maleimide groups [START_REF] Matsumoto | A Water-Based Chitosan-Maleimide Precursor for Bioconjugation: An Example of a Rapid Pathway for an In Situ Injectable Adhesive Gel[END_REF]. Maleimide selectivity and efficient cross-linking with thiol groups, via Michael addition, has allowed the bioconjugation of chitosan with biomolecules such as peptides [START_REF] Martínez-Jothar | Insights into maleimide-thiol conjugation chemistry: Conditions for efficient surface functionalization of nanoparticles for receptor targeting[END_REF] or nucleic acids [START_REF] Malhotra | Development and characterization of chitosan-PEG-TAT nanoparticles for the intracellular delivery of siRNA[END_REF], avoiding the generation of reaction byproducts. In the present work, conjugation between CS and maleimide propionic acid was performed and CS and conjugated CS-maleimide samples were freeze-dried and redispersed in deuterium oxide (D2O).

Conjugation reaction between the polysaccharide and the 3-maleimidopropionic acid group is described in Supplementary Figure 1b. Their H 1 -NMR spectra had been recorded from 0 to about 10 ppm using a proton NMR spectrometer, to confirm the successful conjugation. The typical CS spectrum was confirmed in Figure 2b, which presented a signal at δ ~ 4.9, corresponding to H7 anomeric proton, the ring proton chemical shift (H2-H6) as overlapping signals at δ ~ 3.6-4.0, which confirmed the presence of the glucosamide moiety, while adjacent amino group ring proton (H1) appeared at δ ~ 3.2. The methyl group of the non-deacetylated part of the polysaccharide was expressed at δ ~ 2.1 (NHCOCH3), [START_REF] Kumirska | Application of spectroscopic methods for structural analysis of chitin and chitosan[END_REF], and deacetylation degree >85% could also be confirmed basis of proton integrals. In Figure 2a there still appeared signals similar to that native chitosan signals at 2.1 ppm (NHCOCH3), anomeric proton at 2.9 ppm (H1) and glucosamide moiety at 3.8-4 ppm (H2 to H6) [START_REF] Esquivel | Synthesis and characterization of new thiolated chitosan nanoparticles obtained by ionic gelation method[END_REF]. The presence of new peaks Hb and Hc at ~ 3.5 and 2.7 ppm, respectively, representative of typical protons of the maleimide moiety (Figure 2c), were in accordance with the chitosan chemical modification, proving the successful attachment of chitosan to maleimide propionic acid.

Preparation and physicochemical characterization of chitosan nanocapsules

Physicochemical characterization of nanocapsules

The combination of antigens with adjuvants into an optimal delivery system is a key for the development of a successful vaccine. In addition, aspects such as particle size or surface charge are crucial for the interaction and uptake of particles by immune cells [START_REF] Goulart | Early pneumococcal clearance in mice induced by systemic immunization with recombinant BCG PspA-PdT prime and protein boost correlates with cellular and humoral immune response in bronchoalveolar fluids (BALF). Vaccine X[END_REF]. The delivery carrier was selected based on the knowledge generated by our group and the protein antigen PsaA was associated on the surface of the nanocapsule to obtain a nanovaccine that mimics the pathogen structure and be able to generate a specific immune response.

CS NCs are made of a hydrophobic oily core surrounded by a hydrophilic coating. This core-corona structure uses lipids as core components allowing nanocapsules to interact with and penetrate through mucosal surfaces [START_REF] Alonso | Nanomedicines for overcoming biological barriers[END_REF]. During the first step, we evaluated different ratios of surfactant:oil (data not shown)

and the impact on the particle size and polydispersity of the resulting nanoemulsion (NE), and we selected nanometric size particles with narrow size distribution to form the oily core of the nanocapsules. The amount of Miglyol ® was the main variable affecting the size of nanocapsules. Similar results were previously observed for other polymeric nanocapsule compositions such as those described by González-Aramundiz et al [START_REF] González-Aramundiz | Rational design of protamine nanocapsules as antigen delivery carriers[END_REF]. The use of non-ionic surfactants such as PEG-st 40 helps in preventing aggregation of particles during the formation of initial NE [START_REF] Singh | Nanoemulsion: Concepts, development and applications in drug delivery[END_REF], whilst SGC was used as a co-surfactant that provides negative charge and enhanced stability to the oil core of the NCs [START_REF] Goycoolea | Chitosan-based nanocapsules: Physical characterization, stability in biological media and capsaicin encapsulation[END_REF]. As shown in Figure 3, chitosan molecules were anchored to the surface due to ionic interactions between polymer chains and negatively charged oily core, following an ionic interaction procedure, as previously described for chitosan coating colloidal systems [START_REF] Calvo | Development of positively charged colloidal drug carriers : chitosancoated polyester nanocapsules and submicron-emulsions[END_REF], resulting in a core-coated nanostructure. NCs were prepared using the solvent displacement technique. As shown in Table 1, in all cases, they were monodisperse with a size range of 245-266 nm and the presence of a CS coating surrounding the oily core was corroborated by obtaining a positive surface charge of +30 mV, due to the amine groups in the CS structure. The NCs exhibited an increase in particle size and the surface charge decreased in the presence of antigen when compared with blank CS NCs. This increase in size could be considered as an indicator of a successful binding between the protein and the surface CS of the nanocapsules, and it could also be related to the presence of maleimide-groups as previously described for the development of thiolated-CS liposomes [START_REF] Gradauer | Chemical coupling of thiolated chitosan to preformed liposomes improves mucoadhesive properties[END_REF].

Association efficiency

Nanoparticle formulations prepared with thiol-maleimide coupling resulted in higher PsaA association efficiency than formulations without this covalent modification. Namely, the association efficiency of protein conjugated to the surface of NCs was approximately 3 times higher with PsaA-SATA compared to ones where the antigen was associated without polymer-protein conjugation reaction (86% versus 34%, approximately). Covalently conjugated NCs also preserved their particle integrity and physicochemical properties, so that they were expected to improve antigen delivery and activation of APCs, enhancing immunogenicity compared to non-conjugated NCs.

For uptake studies, the conjugation of CS-maleimide with Cy5 dye was first performed and nanocapsules were prepared using the same procedure previously described. The incorporation of the fluorophore did not alter the NCs original properties. The NTA analysis (Figure 4a) of conjugated CS-maleimide PsaA NCs also confirmed previously reported DLS data showing a particle population of 268 ± 98.5 nm (red line), which corresponds to NCs. Analysis of the conjugated CS NCs in solution showed that the concentration/size distribution was less homogeneous than in case of lyophilized NCs, with more than one population of particles. Smaller NCs could be derived of NTA technique, where a higher dilution of CS-maleimide NCs (1:100) might had occasioned their rupture [START_REF] Maruyama | Nanoparticles Based on Chitosan as Carriers for the Combined Herbicides Imazapic and Imazapyr[END_REF], while aggregation of primary NCs into large NCs could be consequence of the decrease of repulsive forces between the particle surfaces after chemical coating modification [START_REF] Shrestha | Nanoparticle processing: Understanding and controlling aggregation[END_REF].

To assess the stability of conjugated CS-maleimide PsaA NCs after freeze drying process, formulations were lyophilized in the presence of trehalose 5%. After this procedure, the nanocapsules (purple line) were easily redispersed in ultrapure water and a slight change (< 50 nm) in the particle diameter was observed after NTA analysis. It was also observed that a lower concentration of nanocapsules tended to aggregate after lyophilization procedure compared with NCs in solution, which confirmed trehalose as an appropriate cryoprotectant to prevent nanoparticle aggregation. Production yield calculated by the difference obtained between the initial weight and the weight obtained after lyophilization was 67±21%. This freeze-dried chitosan nanocarrier could preserve their integrity upon storage without compromising its physicochemical properties, which is a very attractive feature in terms of developing a future thermostable vaccine formulation [START_REF] González-Aramundiz | Protamine Nanocapsules for the Development of Thermostable Adjuvanted Nanovaccines[END_REF].

Morphological characterization

Different microscopic techniques were used to characterize the conjugated NCs. FESEM and STEM microscopy images revealed a homogeneous population of particles with round-shape structures, which confirm the spherical structure of the NCs. The average particle physical diameter was around 220 nm that corroborated the hydrodynamic NCs diameter values obtained by DLS, where slightly higher values were influenced by the interactions between the particles and the dispersion media (Fig. 4b and4c) [START_REF] Tuoriniemi | Intermethod comparison of the particle size distributions of colloidal silica nanoparticles[END_REF].

Colloidal stability

Apart from obtaining adequate physicochemical properties, particle stability must be guaranteed for the developed nanosystems. The understanding of the colloidal stability and aggregation of the nanosystems in the biological media is important for their rational design. Moreover, the nanoparticle aggregation has shown to influence the in vitro cellular responses. Our studies showed that the size of the CS-maleimide PsaA NCs remained unchanged and no signs of aggregation were observed during a period of 24 h in both RPMI-1640 and SNF in suspension (Figure 5a), and after the freeze-drying process (Figure 5b), which suggest that the NCs were stable in the biological media and could be further proceeded for primary cell culture studies. However, there was a considerable increase in particle size when SNF medium was supplemented with mucin type III. This increase in the particle size of the NCs can be attributed to the decrease in the Brownian motion of the particles due to higher viscosity of the medium in the presence of mucin, as well as structural properties of CS such as deacetylation degree or molecular weight [START_REF] Menchicchi | Structure of Chitosan determines its interactions with mucin[END_REF]. This electrostatic interaction between the positively charged chitosan nanocapsules and the negatively charged mucin molecules yields protein-polysaccharide complexes [START_REF] Collado-González | Interaction between Chitosan and Mucin: Fundamentals and applications[END_REF]. According to Feng et al [START_REF] He | High-throughput dynamic light scattering method for measuring viscosity of concentrated protein solutions[END_REF], dynamic viscosity could be calculated by DLS method, using water viscosity values (~0.89 cP at 25ºC) and commercial polystyrene latex nanoparticles of known size (Magsphere Inc, USA) as reference. Values of ~1.21 cP for SNF media and ~1.41 cP for SNF media supplemented with mucin type III confirmed this increase in dynamic viscosity. Conjugated CS-maleimide PsaA NCs became larger and their brownian movement slower after incubation in SNF with mucin Type III, which might reflect the mucoadhesive properties of chitosan nanocapsules with mucin macromolecules [START_REF] Raskin | Mucoadhesive nanogels by ionotropic crosslinking of chitosan-g -oligo(NiPAam) polymeric micelles as novel drug nanocarriers[END_REF]. Chitosan-mucin interaction by hydrogen and electrostatic bonding has been shown to be suitable for pharmaceutical and biomedical applications in the oral, nasal or ocular routes [START_REF] Szymańska | Stability of chitosan -A challenge for pharmaceutical and biomedical applications[END_REF]. However, future in vivo experiments will be needed to confirm the mucoadhesive properties of CS-PsaA nanocapsules conjugated for nasal application. In addition, all formulations have maintained constant stability profiles in aqueous suspension at 4 °C for at least, 1 month (Supplementary Figure 2).

Release studies

The in vitro release behavior of PsaA from CS NCs was investigated using simulated nasal fluid (SNF pH = 6.5). The accumulative release study of non-conjugated CS-PsaA and conjugated CS-maleimide PsaA NCs was carried out after 24 h of nanocapsules incubation in SNF by analyzing the total amount of free protein in the media at each time point. Total loading was estimated considering the previously reported association efficiency rates achieved after physical (CS-PsaA NCs) and chemical (CS-maleimide PsaA NCs) conjugation. Values of protein released over time were obtained by extrapolation with a BSA protein concentration range in SNF. As observed in Figure 6, NCs presented an initial fast release during the first 3 hours of approximately the half of the PsaA antigen from the formulations to the medium. The release experiment indicated that the protein was gradually released from the nanocapsules in a time-dependent manner, and that this antigen release remained stable after 8 h. However, a sustained antigen release pattern was observed after 3 hours in the CS-PsaA NCs, reaching values of 100% of the release at 24 h, contrary to the CS-maleimide NCs, where the release was slowed down, reaching values over 76% at 24 h. The relatively slower and sustained release in conjugated CS-maleimide PsaA formulation may be ascribed to the chemical conjugation which produced a stronger interaction between the antigen and the polysaccharide. As showed in similar studies in maleimide-bearing nanogels [START_REF] Tonglairoum | Maleimide-bearing nanogels as novel mucoadhesive materials for drug delivery[END_REF] and maleimidefunctionalized PEGylated liposomes [START_REF] Kaldybekov | Mucoadhesive maleimidefunctionalised liposomes for drug delivery to urinary bladder[END_REF], carrier capacity to retain and release the drug was sustained for a prolonged time-period. Overall, both conjugated and free antigen release presented a biphasic profile with a faster initial release followed by a second phase with slow and delayed tendency release profile.

NCs toxicity on immune cells

To investigate the biocompatibility of both types of conjugated CS-maleimide nanocapsules (blank and antigen loaded), an in vitro MTS assay was performed to study the influence of the nanocapsules on the metabolic activity of macrophages and DCs. Since cationic nanoparticles generally have better electrostatic attractions with the negatively charged cell surface, their toxicity also might increase [START_REF] Tzankova | In vitro and in vivo toxicity evaluation of cationic PDMAEMA-PCL-PDMAEMA micelles as a carrier of curcumin[END_REF]. After 4 h (Figure 7a) and 24 h (Figure 7b) of CS-maleimide and CS-maleimide PsaA NCs incubation with macrophage Raw 264.7 cells, MTS assay showed a significant reduction in cell viability (mitochondrial reductase activity) at concentrations > 200 µg/mL. Membrane damage was more pronounced in blank CS-maleimide NCs, being practically total at values of 800 µg/mL, while in the case of protein-conjugated nanocapsules it remained around 40% at higher doses. Similar effects on toxicity were observed after incubation of NCs with iDCs for 24 h (Figure 7c), although in this case, toxicity was apparently higher in blank CS-maleimide NCs.

In a second instance, cell toxicity values were also verified via 7-AAD cytotoxicity assay test at 24 h.

Considering that dendritic cells with compromised membranes were stained with 7-AAD, untreated cells were use as control for viable cells. The flow cytometry analyses performed (Supplementary Figure 3)

showed a decrease in the number of viable cells as the CS-maleimide PsaA NCs concentration increased, reaching dendritic cell viability values of 65% at NCs concentrations of 900 µg/mL. Chitosan; NCs: Nanocapsules.

Interaction of NCs with immune cells

Immune cells are able to capture antigens in many different ways, such as phagocytosis, macropinocytosis, or by expressing surface receptors causing endocytosis [START_REF] Mellman | Minireview Dendritic Cells: Specialized and Regulated Antigen Processing Machines cells are adept at endocytosis and express relatively low levels of surface MHC class I and II products and costimu-latory molecules (e.g., CD86). Abundant MHC class II mol[END_REF]. Once the antigen has been captured by the antigen presenting cells, it is processed and presented on the cell surface by Major Histocompatibility

To evaluate the effect that NCs may have on iDC phenotype, the expression of T lymphocyte costimulatory molecules CD83 and CD86 (indicators of pro-inflammatory activated phenotype in dendritic cells) were analyzed by flow cytometry. The co-stimulatory marker CD86 designates to be a marker primary DCs maturation expressed within first 24 h and interacts with the CD28 for the T cell activation, while the increased CD83 is observed in mature/activated DCs. Several studies have proven that antigen delivery to DCs upregulates expression of both CD83 and CD86 that are known to induce T cell receptor signalling and promote T cell activation [START_REF] Gmz | Dendritic Cell Subsets, Maturation and Function[END_REF]. In our study, both the blank and antigen loaded CS-maleimide NCs showed an increase in the expression of these maturation markers. The expression of CD83 and CD86

(Figure 9a) were 4 folds higher in the case of DCs primed with blank and antigen loaded CS-maleimide NCs when compared to iDCs (MFI=1). However, iDCs treated with PsaA alone displayed a 2 and 3-fold lower expression of CD83 and CD86 markers, respectively. Overall, the results suggest that either the blank or antigen loaded NCs can upregulate the co-stimulatory markers in comparison to the antigen alone.

NCs tolerogenic effect on immature DCs

Antigen-presenting cells such as iDCs play an important role in immune regulation, not only as inductors but also inhibitors of an immune response. 2,3-Indoleamine dioxygenase (IDO) is an enzyme involved in tryptophan catabolism, essential for the growth of microorganisms, which regulates T-cell tolerance [START_REF] Braun | A two-step induction of indoleamine 2,3 dioxygenase (IDO) activity during dendritic-cell maturation[END_REF].

IDO is a potent immune-suppressive enzyme in macrophages and dendritic cells whose physiological function is to catabolize the essential amino acid tryptophan into the stable metabolite, kynurenine which is known to induce the apoptosis of Th1 cells in vitro [START_REF] Fallarino | T cell apoptosis by tryptophan catabolism[END_REF]. In addition, it is known that the IDO expression by the DCs can greatly impact peripheral tolerance and immune regulation. In this line, we performed a study to evaluate if the NCs formulations were inducing a tolerogenic phenotype on iDCs in terms of Tcell suppression and tolerance promotion (opposite to a pro-inflammatory response), by quantifying the IDO enzymatic activity after iDCs treatment with NCs. The results obtained from our study showed that the response in the DCs treated with either blank or antigen loaded CS-maleimide NCs signal was similar to one observed in iDCs (Figure 9b), indicating no tolerogenic effect of the NCs. In all cases, incubation of iDCs in the presence of NCs caused these APCs to have a similar capacity to stimulate CD4 and CD8 lymphocytes to iDCs, and were lower compared to mDCs. Also, levels of both activated CD4+CD25+ and CD8+CD28+ CD4 T cells were similar for loaded and non-loaded nanocapsules. However, no significant differences were found between treatments, probably due to donor variability. The similar values between both treatments on the effect of T cell activation could be a consequence of the composition of the nanocapsules, rather than the presence of the antigen on the surface. [START_REF] Caulfield | Information about subscribing to The[END_REF]. In the process of developing a powerful immune response, dendritic cells capture antigens thanks to pattern recognition receptors (PRRs), leading to phenotypic and maturation changes [START_REF] Craig | Neutrophil recruitment to the lungs during bacterial pneumonia[END_REF]. Depending on the alterations they undergo and the co-stimulation molecules they express, different responses will take place from the T cells (Th1, Th2 or Th17) [START_REF] Dzopalic | The response of human dendritic cells to co-ligation of pattern-recognition receptors[END_REF]. The secretion of cytokines by Th1 CD4+ cells (such as IL-2, IFNɣ, IL-10 or TNFα) is necessary in order to achieve protection against pneumococcal infection [START_REF] Singh | Prediction and characterization of helper T-cell epitopes from pneumococcal surface adhesin A[END_REF].

TNFα, secreted by either macrophages, monocytes or activated lymphocytes acts as an inflammatory marker and protects against the infectious pathogens. TNFα production can activate the T cells or can be a product of the activated T cells. In this procedure, we quantified by intracellular cytokine staining the production of TNF by T lymphocytes (CD3+) and non-T cells (CD3-) after incubation with NCs. As can be seen in Figure 11, TNFα production was higher in the CD3-(monocytes) population, being around 5-8% compared to CD3+ (lymphocytes) population, where it did not exceed a 5% in any case. In both cases, a higher percentage of TNF-positive cells was seen after incubated with the different NCs and the PsaA protein compared to iDCs, especially for the CD3+ T cells. Although no statistical differences were found in the different treatments due to the high variability among donors, the obtention of slightly higher cytokine secretion levels in CS-maleimide PsaA NCs could be a consequence of the presence of PsaA attached on their surface. These results were in agreement with previous studies evaluating the effect of cationic CS NCs in inducing inflammatory responses, where cytokine levels were remarkable in one out of three donors tested [START_REF] Peleteiro | Polymeric nanocapsules for vaccine delivery: Influence of the polymeric shell on the interaction with the immune system[END_REF]. Likewise, when the PsaA protein was used, a clear increase in the percentage of TNF-positive cells was observed when compared to control cultures. This may indicate the external presence of the bacterial endotoxin lipopolysaccharide LPS, a potent activator of innate immunity.

Conclusion

In the design of targeted nanovaccines towards nasal immune cells, a specific nanocarrier design with mucoadhesive properties is crucial to achieve a higher residence time, avoiding mucociliary clearance and crossing nasal epithelium. In this work, combination of the antigenic protein membrane PsaA together with the high capacity of drug association of chitosan-maleimide modified nanocapsules has been positioned as an interesting perspective for Streptococcus pneumoniae vaccination. Results supported the versatility of non-capsular pneumococcal antigens as vaccine candidates for an efficient mucosal administration.

Developed nanocapsules presented a high effective payload of antigen after covalent thiol linkage, mucoadhesiveness together with high physical and biological stability. The nanocapsules capacity to interact with immune cells was conserved and enhanced compared to the free antigen, suggesting them a "green light" future serotype-independent pneumococcal vaccine candidate. To further assess the ability of these nanocapsules as vaccine delivery systems, the evaluation of antigen-specific responses and cytokine profiles is currently under way.

Conflicts of interest/Competing interests

Authors declare that they have no conflict of interest.

1 -

 1 Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, C8H17N3, Mw: 191.7 Da), N-Hydroxysuccinimide (NHS, C4H5NO3, Mw: 115.09 Da), sodium chloride (NaCl), potassium chloride (KCl), calcium chloride hydrate (CaCl2•2H2O) and mucin type III were purchased from Sigma Aldrich (MO, USA). Maleimide propionic acid 95% (m-PA, C11H10N2O6, Mw:169.13 Da) and hydroxylamine were obtained from Alfa Aesar (MA, USA). Cyanine5 NHS ester dye (Cy5-NHS) was provided by Lumiprobe (Hannover, Germany), S-acetylthioacetate succinimidyl ester (SATA, Mw: 231.23 Da) and the SnakeSkin TM Dialysis Tubing 3.5K MWCO were obtained from Fischer scientific (MO, USA). The Ni-NTA resin was obtained from Macherey-Nagel, and PsaA used in the experiments was provided by UFIP UMR CNRS 6286 at University of Nantes. For in vitro experiments sterile and autoclaved material were used. Macrophage Raw 264.7 cell line was obtained from ATCC (VA, USA), Ficoll-Paque™ PLUS (density 1.077 g/mL) was purchased from GE Healthcare Bio-Science AB (IL, USA), Roswell Park Memorial Institute medium (RPMI-1640) and Dulbecco's Modified Eagle's Medium (DMEM) were obtained from GIBCO ® (Thermo Fisher Scientific, MA, USA). Granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), allophycocyanin (APC)-conjugated anti-human CD83 (CD83-APC), phycoerythrin (PE)-conjugated antihuman CD86 (CD86-PE) and fluorescein (FITC)-conjugated anti-human CD1a (CD1a-FITC), were purchased from Miltenyi Biotec (CA, USA), anti-human CD4-APC, CD8-APC; CD25-PE and CD28-PE were obtained from Tonbo Biosciences (CA, USA). Fluorescein (FITC)-conjugated anti-human CD3 (CD3-FITC) and phycoerythrin (PE)-conjugated anti-human Tumor Necrosis Factor alpha (TNF-) were both from Miltenyi Biotec (Madrid, Spain). Fetal Bovine Serum (FBS), PSG (100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM L-glutamine) and Ebioscience TM 7-aminoactinomycin D (7-AAD) viability staining were purchased from Invitrogen (CA, USA). Lipopolysaccharide (LPS) from Escherichia coli and Brefeldin A (BFA) were purchased from Sigma Aldrich (MO, USA). MTS (reactive de Owen (3-(4,5dinetiltiazol-2-il)-5(3-carboximetoxifenil)-2-(4-sulfofenil)-2H tetrazolium) cell proliferation assay kit was

Fig. 1 :

 1 Fig. 1: Pre-formulation characterization of PsaA and its conjugate PsaA-SATA: (a) graphs of MALDI-TOF for PsaA (blue line) and PsaA-SATA conjugate (green line). (b) Overlay of CD spectra for PsaA and

Fig. 2 :

 2 Fig. 2: H 1 -NMR (400 MHz, D2O) spectra and integration peaks of Chitosan-maleimide (a), Chitosan (b), 3-maleimidopropionic acid (c), and their respective chemical structures (right). All chemical shifts were expressed as δ in parts per million (ppm), using the chemical shift of the residual solvent resonances as references (D2O: δ = 4.80 ppm).

Fig. 3 :

 3 Fig. 3: Schematic representation of chitosan polymeric NCs developed in this work. Non-conjugated CSantigen nanocapsules (left) and CS-antigen nanocapsules after thiol/maleimide conjugation (right). Design was created by Biorender ® and ChemDraw ® graphics programs.

Table 1 :

 1 Physicochemical characterization and association efficiency of antigen-conjugated and nonconjugated NCs. Particle size and polydispersity index were obtained by Dynamic Light Scattering. Data represent mean ± S.D., n=8. CS: Chitosan; NCs: Nanocapsules; PDI: Polydispersity index.

Figure 4 :

 4 Figure 4: (a) Representative image of the particles size distribution by Nanoparticle Tracking Analysis of conjugated nanocapsules in solution (red line) and conjugated nanocapsules resuspended after lyophilization procedure (purple line), with the mean particle size ± standard deviation. Electron microscopy scanning (b) and transmission (c) images of conjugated Chitosan-maleimide PsaA NCs. Scale bars are indicated in each image. NCs: Nanocapsules.

Fig. 5 :

 5 Fig. 5: Stability of conjugated CS-maleimide PsaA NCs in suspension (a) and lyophilized (b) conditions in water, cellular media (RPMI), simulated nasal fluid alone (SNF) and with mucine type III (SNF+ 0.1% Mucin). Bars represent size (nm), and dots represent derived count rate (kcps) (mean± SD; n=3).

Fig. 6 :

 6 Fig. 6: In vitro cumulative release profile of PsaA antigen from chitosan PsaA and chitosan-maleimide PsaA NCs (mean± S.D.; n=6). CS: Chitosan, NCs: Nanocapsules.

Fig. 7 :

 7 Fig. 7: Effect of CS-maleimide and CS-maleimide PsaA NCs on the viability of Raw 264.7 macrophages (a, b) and immature dendritic cells (c). MTS assay performed on Raw 264.7 cell lines incubated with either CS-maleimide (blue lines) or CS-maleimide PsaA (red lines) NCs using different concentrations of nanocapsules during (a) 4 h and (b) 24 h. (c) MTS assay performed on iDCs after incubating them with different concentrations of nanocapsules for 24 h. Results were normalized with the percentage of live iDCs incubated in culture media alone and dead iDCs incubated with Triton (5%, 1x) (mean ± S.D; n=4). CS:

Fig. 9 :

 9 Fig. 9: (a) Quantification (%) of the expression of iDC maturation markers CD83 and CD86. Results are expressed as the ratio between the markers mean fluorescence intensity (MFI) in iDCs incubated with the different NCs and the MFI of iDC incubated in culture media alone (MFI=1). The dotted line corresponds to the signal from the iDCs. (b) Indoleamine 2,3-dioxygenase (IDO) activity in dendritic cell cultures. iDCs: immature dendritic cells; CS: Chitosan; NCs: Nanocapsules) (mean ± SEM; n=3 donors).

Fig. 10 :

 10 Fig. 10: Percentages (%) of activated T cells after iDCs incubation with formulations. Non-treated iDCs and LPS and IFNɣ treated iDCs (mDCs) were used as controls (mean ± SEM; n=3 donors, 2-way ANOVA test). CS: Chitosan; iDCs: immature Dendritic Cells; mDCs: mature Dendritic Cells; NCs: Nanocapsules.

Fig. 11 :

 11 Fig. 11: Percentages (%) of TNFα positive cells (CD3+ and CD3-) after treatment with CS-maleimide NCs, antigen loaded CS-maleimide PsaA NCs and PsaA antigen alone (mean ± SEM; n=3 donors, 2-way ANOVA test). CS: Chitosan; NCs: Nanocapsules.

the

  Xunta de Galicia (Centro Singular de Investigación de Galicia accreditation 2019-2022) and the European Union (European Regional Development Fund -ERDF), code ED431G 2019/02. Maruthi Prasanna acknowledges his doctoral fellowship from the European Commission, Education, Audiovisual and Culture Executive Agency (EACEA), under the Erasmus Mundus program, "NanoFar: European Doctorate in Nanomedicine and Pharmaceutical Innovation" (Project: 2015-01-C4). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Acknowledgment & Funding

This work was supported by 'Ministerio de Economía y Competitividad' -(RETOS -SAF2016-79230-R -ERC2018-092841 and PID2019-108727RB-I00). Sandra Robla acknowledges the financial support from

Availability of data and material

Not applicable

Code availability

Not applicable

Complex (MHC) [START_REF] Banchereau | Dendritic cells and the control of immunity[END_REF]. To confirm whether our nanocarriers were able to interact with such immune cells, we examined the efficacy of CS-maleimide PsaA NCs on antigen uptake in primary cultures of human monocyte-derived iDCs and in the macrophage Raw 264.7 cell line. After incubating the Cy5-labelled NCs (both blank and antigen loaded), the presence of intracellular fluorescence due to the Cy5-labelled NCs confirmed the internalization of the nanocapsules by the DCs. The images obtained from the confocal scanning microscopy confirmed that the nanocapsules (top Figure 8a, red channel) were located within the dendritic cells (bottom, contrast microscope DiC channel) and intracellular distribution of CS-maleimide NCs could be seen throughout the different sections of dendritic cells (also see online video resource in Supplementary material). The physical appearance of DCs could also provide information about their maturation and activation status, for example, by the formation of their characteristic dendrites as seen in our case [START_REF] Kowalewicz-Kulbat | Type of monocyte immunomagnetic separation affects the morphology of monocyte-derived dendritic cells, as investigated by scanning electron microscopy[END_REF].

On the other hand, the interaction of macrophages with nanocapsules was analyzed by FESEM microscopy.

The presence of protrusions on the surface of cells observed in FESEM images (Figure 8c) suggested a possible grouping of nanocapsules and recognition by macrophages. to ease their visualization.

Human DCs phenotype analysis

Ethics approval

All procedures followed were in accordance with the ethical standards of the responsible institutional and national committees on human experimentation and with the Declaration of Helsinki. Informed consent was obtained from all patients for being included in the study.

Consent to participate

All institutional and national guidelines for the obtention and use of human blood were followed. Blood was drawn with the informed consent of all subjects and appropriate permission was obtained from the Institutional Ethics Committee (Comité Etico de Investigación Clínica de Galicia, CEIC).

No animal or human studies were carried out by the authors for this article.

Consent for publication

Not applicable

Authors' contributions 28 Sandra Robla was in charge of the overall experimental execution, data analysis, writing and figure preparation. Maruthi Prasanna contributed to the experimental execution and writing. Cyrille Grandjean was involved in the synthesis and characterization of the PsaA antigen, reviewing and editing the manuscript. R Varela-Calvino was involved in the design and supervision of the in vitro cell experiments, reviewing and editing the manuscript. Noemi Csaba was involved in writing, reviewing, editing, supervision, project design and administration.

Supplementary material

Supplementary Figure 1: (a


Mohammadreza Nezamirad
email: mnezamirad@usf.edu

Nasim Sabetpour

Azadeh Yazdi

Amirmasoud Hamedi
email: amirmasoud.hamedi1@fiu.edu

Investigation the Effect of Velocity inlet and Carrying Fluid on the Flow inside Coronary Artery

Keywords: CFD, Heart simulation, OpenFOAM

In this study OpenFOAM 4.4.2 was used to investigate flow inside the coronary artery of the heart. This step is the first step of our future project, which is to include conjugate heat transfer of the heart with three main coronary arteries. Three different velocities were used as inlet boundary conditions to see the effect of velocity increase on velocity, pressure, and wall shear of coronary artery. Also, three different fluids namely the University of Wisconsin solution, Gelatin, and blood was used to investigate the effect of different fluids on flow inside coronary artery. A code based on Reynolds Stress Navier Stokes (RANS) equations was written and was implemented with the real boundary condition that was calculated based on MRI images. In order to improve the accuracy of the current numerical scheme, hex dominant mesh is utilized. When the inlet velocity increases to 0.5 m/s, velocity, wall shear stress, and pressure increase at the narrower parts.

Introduction

Modeling and simulation of heart is a very complex phenomena which can take significant amount of time and research facilities. One of the biggest problems that nowadays donors and recipients are dealing with is the vast geographical differences that can make the process of compatible transplantation very hard and in many cases impossible [START_REF] Kannojiya | Simulation of Blood as Fluid: A Review From Rheological Aspects[END_REF][START_REF] Siamak | The numerical analysis of non-Newtonian blood flow in human patient-specific left ventricle[END_REF]. In large arteries, shear stress can be exerted linearly [START_REF] Collins | Preserving and Evaluating Hearts with ex vivo Machine Perfusion: an Avenue to Improve Early Graft Performance and Expand the Donor[END_REF]. A model for design of cardiovascular devices by acquisition of COMSOL Multiphysics software was introduced [START_REF] Garry | A MULTIPHYSICS SIMULATION OF A CORONARY ARTERY[END_REF]. Distention and wall shear stress were used to validate the mentioned model. Non-Newtonian blood flow was simulated by acquisition of laminar module in order to investigate lateral angle (LA) ratio main middle cerebral artery (MCDA) which can be correlated to aneurysm formation. It was found that varying MCDA bifurcation angles can increase aneurism due to wall shear stress and changes the fluid flow. Patient specific modeling for measuring wall shear stress and hemodynamic in coronary arteries with aneurism which was caused by Kawaski Desease (KD) was investigated by Sengupta et al. [START_REF] Luis | ANALYSIS OF PARTICLES THOROUGH THE AORTIC ARCH DURING TRANSCATHETER AORTIC VALVE REPLACEMENT[END_REF]. In the current study, since our next purpose is heart cooling for transplant purposes, Gelatin and UW solutions was investigated beside blood for flow inside coronary artery. The human heart geometry used for simulations was obtained from three-dimensional, high resolution MRI scans. Figure 1 demonstrates different domain surfaces of the heart in different colors. Figure 1 shows a complex model of heart which has three coronary arteries. According to figure 1a, full heart model with three coronary arteries can be seen. Figure 1b shows three coronary arteries which are located in their correct places. The model which is shown in figure 1 was obtained from MRI image from University of Minnesota. Figure 1c shows coronary artery which will be simulated in this study using Figure 2. Mesh used for coronary artery which was used for the present study.

Result and discussion

Figure 3 shows pressure distribution when UW solution was used. Figure 3a shows pressure distribution inside coronary artery when inlet velocity is 0.2 m/s and outlet velocity is zero. Figure 3b shows pressure distribution when inlet velocity is 0.35 m/s and outlet velocity is zero. Figure 3c Figure 6 shows pressure inside coronary artery when inlet velocity is 0.2 m/s and outlet velocity is zero. For case a in figure 6, UW solution was used and as mentioned before pressure reaches to 8.79 Pa. By comparing figure 6a, 6b and 6c, it can be found that since Gelatin has the greatest amount of kinematic viscosity ( ), the biggest amount of peak pressure (10.15 Pa) also occurs in this case. Blood due to lowest amount of kinematic viscosity ( ) has the lowest amount of peak pressure (7.96 Pa). In OpenFOAM, the unit of wall shear stress is . Figure 8 shows distribution of wall shear stress when three different fluids were used. Among the three different cases which are shown in figure 8, case a which is UW solution has the highest amount of wall shear stress but it only occurs in few regions of coronary artery. Although using Gelatin (case b) can be a reason for low value of maximum wall shear stress but in some regions of case b high values of wall shear stress is noticeable.

World Academy of Science, Engineering and Technology International Journal of Aerospace and Mechanical Engineering Vol: 15, No:07, 2021 Figure 8. Magnitude of wall shear stress inside coronary artery when three different solutions were used and inlet velocity was 0.2 m/s (case a. UW solution, case b. Gelatin, case c. blood).

Conclusion

In this study, OpenFOAM 4.4.2 open source software was used to simulate flow inside coronary artery. SimpleFOAM solver was used to solve Reynolds Average Navier Stoks (RANS) equations. It was found that as inlet velocity increases inside coronary artery, pressure, velocity and wall shear stress increases when the carrying fluid is UW. Also, three different carrying fluids was used in order to investigate the effect of different fluids on velocity, pressure and wall shear stress distribution and it was found that using Gelatin can cause higher value of peak velocity and pressure. Amazingly the highest value of peak pressure was for UW solution but it was occurred in few regions. Although the lowest value of wall shear stress was for Gelatin, maximum value of wall shear stress occurred in numerous regions.

Figure 1 .

 1 Figure 1. The whole heart geometry with three coronary arteries inside it.

Figure 2

 2 Figure2shows the mesh which was used for the present study in order to investigate flow inside coronary artery. Figure2ais the whole geometry of coronary artery. Figure2bshows close up view for the inlet of coronary artery with its mesh. In this study, three different boundary conditions for three different fluids were used. At first it was assumed that inlet velocity is 0.2 (m/s) and then inlet velocity was changed to 0.35 (m/s) and 0.5 (m/s). In all of the cases outlet velocity was set to zero. Three different fluid that was used in this study are orderly, University of Wisconsin (UW) solution, Gelatin and blood.

Figure 3 .

 3 Figure3shows pressure distribution when UW solution was used. Figure3ashows pressure distribution inside coronary artery when inlet velocity is 0.2 m/s and outlet velocity is zero. Figure3bshows pressure distribution when inlet velocity is 0.35 m/s and outlet velocity is zero. Figure3cshows velocity distribution when inlet velocity is 0.35 m/s and outlet velocity is zero. According to figure3, increasing velocity from 0.2 m/s to 0.35 m/s, increases pressure from 8.79 Pa to 20.2 Pa which is nearly 130 percent.

Figure 4 Figure 4 .

 44 Figure4shows velocity distribution along coronary artery when the flowing fluid is UW. Three different cases were investigated. In case 1, inlet velocity was 0.2 m/s and outlet velocity were set to zero. In case 2 inlet velocity was 0.35 m/s and outlet velocity were zero m/s. Finally, in case 3, inlet velocity was 0.5 m/s and outlet velocity were zero. From figure4, it can be inferred that as velocity inlet is 0.2 m/s, the maximum velocity inside coronary artery reaches to 2.49 m/s. As inlet velocity increases to 0.35 m/s, maximum velocity reaches to 4.36 m/s. Finally, when inlet velocity increases to 0.5 m/s, maximum velocity reaches to 6.2 m/s. Maximum velocity in figure4can mostly be observed in the regions that diameter of coronary decreases rapidly.

Figure 5

 5 Figure5shows magnitude of wall shear stress as different inlet velocities were used to evaluate flow field in coronary artery. As figure5ashows, when inlet velocity is 0.2 m/s, maximum value of wall shear stress mostly forms toward outlet of coronary artery and it reaches to its peak value which is 0.236 ( ). After changing inlet velocity to 0.35 m/s, wall shear stress inside coronary artery increases to 0.462 ( ) and it

Figure 6 .

 6 Figure 6. Pressure inside coronary artery when three different solutions was used and inlet velocity was 0.2 m/s (case a. UW solution, case b. Gelatin, case c. blood).

Figure 7 Figure 7 .

 77 Figure 7 shows velocity distribution inside coronary artery when three different fluids were used. Since difference in kinematic viscosity cannot affect velocity significantly, velocity inside coronary artery as kinematic viscosity changes does not change noticeably.


Peshal Nayak

A Study of Technology Roadmap for Application-Specific Integrated Circuit

The International Technology Roadmap for Semiconductors (ITRS) designs roadmap for the researchers and engineers in the ASIC technology industry every few years. This group is divided into seven focus teams. In this paper, I review the future goals and challenges provided by the Systems Integration, Outside System Connectivity and the Factory Integration group 1 .

INTRODUCTION

In order to ensure a systematic and rapid growth in the ASIC technology industry, there is a need to analyze the demands from consumer end and create short term higher level goals for researchers and engineers in the industry. To this end, the Semiconductor Industry Association (SIA) has taken up the challenge to create these roadmaps every few years. Consequently the International Technology Roadmap for Semiconductors (ITRS) which is sponsored by industry leaders in five countries including Europe, Japan, Korea, Taiwan and United States proposes challenges and goals that need to be met to ensure the desired level of progress.

The ITRS is divided into 7 teams namely System Integration, Heterogeneous Integration, Heterogeneous Components, Outside System Connectivity, More Moore, Beyond CMOS and Factory Integration. While these groups have their own targets and activities, there is an interdependence between these groups. This interdependence is as shown in fig. 1.

In this report, I review the literature provided by the Systems Integration, Outside System Connectivity and Factory Integration groups. These groups seem to be closely related to the area of systems research. Since I specialize in wireless systems and networks, the work done by these groups is very relevant to my research which is the main reason for choosing them.

In section 2, review of the works of system integration group is provided. Section 3 describes a review of outside system connectivity while Section 4 studies Factory Integration. References are provided in section

SYSTEM INTEGRATION

The objective of this group is to identify the trends in system products that drive ITRS technologies. The target systems are smartphones, datacenters and microservers and IoT smart objects. The prime research direction involves extraction and analysis of data, identification of performance metrics, using models to understand issues, and identifying areas that need immediate investment to ensure smooth progress in the future.

To do so they divide their work into the following sections:

1) Smartphones: For smartphones, the group identifies image processing, sensing and data analysis and compact integration of devices into a package as the demands of the future obtained from an analysis of the present market trends. Specifically, they highlight the need for research and development in computer vision, data mining and MEMS integration as the key requirements. They highlight the fact that with the advent of wearables, there is little clarity on how different functions will get divided into different system components.

The key technologies that are driven by advancement in the smartphone domain are those related to system functionalities(e.g. number of cores in APs and GPUs), lithography, integration and packaging and those associated with advanced interconnect.

The group also identifies that the form factor for a smartphone architecture, the power consumption, increasing demand for high data rate and higher resolution in sensing hardware as the key challenges in the near future. It is interesting to note that while the group recognizes increasing bandwidth demands in cellular and wifi domains, it completely ignores new physical layer(PHY) schemes such as MU-MIMO [7-11, 13-16, 25] which not only demand advanced PHY with channel state based precoding capabilities different from those of omnidirectional single antenna systems [1, 12, 17-23, 23, 24]. Also, the new 3.5 GHz band that is released by Federal Communications Commission(FCC) for innovation and research (which would also change the challenges at the PHY) has not been addressed [2]. Based on their analysis, this group predicts two system level architecture: one targeted for 2019 (fig. 2) and one beyond 2020 (fig. 3). It also interesting to note in that upcoming technologies such as visible light communications (VLC) have not been addressed even in the architecture beyond 2020.

Figure 2: Smartphone architecture predicted for 2019 [START_REF] Kahng | system integration focus team presentation[END_REF] 2) Datacenter and Microserver: This section focuses on the technologies that are required to interconnect different components in data centers and microservers. The group predicts that in the future, the electrical cables that connect different racks in a data center would be replaced by optical cables. There is also a possibility that they may be replaced by wireless point-to-point links operating in the 60 GHz band which has been ignored as well [START_REF]Intel news on 60GHz[END_REF].

The group identifies that the same technologies that are driven by the smartphone development are also driven by the growing demands in the datacenter and microserver domain. The group predicts a near exponential growth in the demand for storage capacity, interconnect bandwidth and the computation capabilities at the data cen-ter end while a linear growth in the power demands. The group predicts a similar trend for microservers as well. The predicted architecture for microservers beyond 2020 is as shown in fig. 4. Just like the trends for data centres, the group predicts that the number of MPU cores and MPU frequencies will increase almost exponentially until 2019 while the maximum frequency will increase linearly. 3) IoT smart object: This section deals with connectivity, sensing, energy and security related issues for the Internet of Things components. The major concern here is power consumption. The group believes that the system performance must be increased while meeting power constraints on the system. The group shows a trend in the lowest system voltage and predicts a drop by a factor of 4 in 2017 under the assumption that energy harvesting is introduced in that year. This appears to be a very ambitious assumption for IoT given the large variety of devices it spans. The group identifies form factor and system level power as the key challenges. However, one of the main challenges in IoT is device level security and encryption since it involves a large number of wearable devices. This issue has not been fully addressed here.

OUTSIDE SYSTEM CONNECTIVITY

The outside system connectivity group focuses on the requirements and challenges involved in connecting devices involved in a system. The primary objective of this group is to enable efficient communication capabilities amongst the system components. For the next five years, they intend to focus on the Internet of Everything (IoT) and cloud communications. This involves filling the technology gaps required to interconnect the devices present in these two systems, including but not limited to sensors, that are useful for the areas of mobility, energy, health, etc.

To develop novel solutions, the group divides their work into two prime areas: To explain the future directions, the group highlights the roadmaps for frequency, minimum noise figure and associated gain. These are shown in fig. 5, 6 and7. An interesting thing to note here is the trend in the curves. These curves seem to show an abrupt jump every two years and then a flat behavior for the next two years. These jumps can be attributed to the fact that this group deals with device level properties associated with connectivity. It takes a finite period before existing devices reach saturation in terms of the capabilities that they provide thereby paving way for better and advanced technology to boost the device capacities.

2) Optical interconnect applications: The main objective here is to enable efficient connectivity in data centers, telecommunications, fibre optic to X (FTTX: where X could be home, office, curb, etc), automotive and aerospace. It is interesting to note that the group does not intend to focus on health care for the next few years. The reasons are not clear. The prime goal in all of these areas is to enable high speed inter-component communication. Some areas have additional demands that need to be met. For instance, automotives require optimization of the weights of the components involved while aerospace systems require additional focus on electromagnetic interference(EMI) reduction as it could hamper the communication between the system and the ground station, immunity to high temperatures and mechanical shocks involved in the system operation.

The group identifies challenges that it faces in the area of component integration packaging. Specific examples include lasers, modulators and compact waveguides. The group also provides other examples. It seems that the group has been solving some of the problem that might fall under the heterogeneous integration group. This might be due to a lack of co-ordination between these two groups.

Other challenges involve optical switching and routing and deisgn of components to enable amplification and regeneration of optical signals.

For enhancing the quality of communication, the system identifies the performance meterics that include bandwidth, power, form factor and other features required by the international technology working group (iTWGs). The group also specifies the bottleneck elements involved in improving connectivity. These lasers, input couplers, fiber, output coupler, detectors, etc.

The group has made progress in area of simulation, modelling and testing in terms of identifying the key components that need to be studied and tested. For modelling and simulation, the group believes that the main focus should be on design optimization of nanophotonic lasers, routing and plasmonic structures. For testing, the group intends to focus on optical interconnects. Broad testing areas include laser and detector test, alignment requirements, wafer level detectors and emitters, number of fibre and wavelengths invovled in photonic elements.

It is interesting to note that for simulation, modelling and testing the group intends to focus on the features associated with the physical layer (PHY). While an efficient PHY is required to enable high speed communication, the actual data exchange happens at higher layers. This implies that the performance of the system also crucially depends on layers running on top of the MAC. The bottlenecks and performance metrics in these layers seem to be entirely ignored by the group.

FACTORY INTEGRATION

The focus of this group [START_REF] Moyne | factory integration presentation[END_REF] is on the tools and processes that can optimize the cost per volume of the products manufactured. The goal is to make the process of mass manufacturing of goods efficient and environmental friendly. To this end, this group has added Environmental Safety and Health (ESH) and Yield Enhancement (YE) sections to its activities. The main additions that were proposed for 2015 included those related to ESH, YI, Factory operations and security and supply chain integration. With the development of several environmental issues, the ESH addition seems a justified consideration for the future work of this group. However, it is interesting to note that the work from past years which focused on data analysis and control systems seemed to have overlooked this area.

For challenges, the group highlights that the areas of 2-D and 3-D materials and security lacks resources. Especially on the security side, it mentions the lack of expertise on team. While a significant efforts is seen in the area of data analytics, the exact role played by a factory integration group in this area is unclear. In the area of ESH, the group specifically highlights the danger of SVHC(substance of very high concern) compound lists from Taiwan and stresses the need to involve green chemistry principles in the manufacturing of materials.

CONCLUSION

In this paper a review of the future goals and challenges provided by the System Integration, Outside System Connectivity and the Factory Integration group of the ITRS is provided.

Figure 1 :

 1 Figure1: Focus teams in the ITRS and their dependencies[START_REF] Kahng | system integration focus team presentation[END_REF]

Figure 3 :

 3 Figure3: Smartphone predicted beyond 2020[START_REF] Kahng | system integration focus team presentation[END_REF]

Figure 4 :

 4 Figure4: Microserver architecture predicted beyond 2020[START_REF] Kahng | system integration focus team presentation[END_REF]

Figure 5 :

 5 Figure5: Roadmap for frequency requirements until the production year 2028[START_REF] Garner | outside system connectivity presentation[END_REF]

Figure 6 :

 6 Figure 6: Roadmap for minimum noise figure requirements until the production year 2028 [3]

Figure 7 :

 7 Figure 7: Roadmap for associated gain until the production year 2028 [3]