Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan

Journal Pre-proofs Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan Selin Yüksel, Mert Pekcan, Nuhan Puralı, Güneş Esendağlı, Ece Tavukçuoğlu, Vanessa Rivero-Arredondo, Luis Ontiveros-Padilla, Constantino López-Macías, Sevda Şenel PII: DOI: Reference:

S0378-5173(20)30113-7 https://doi.org/10.1016/j.ijpharm.2020.119129 IJP 119129

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

2 December 2019 7 February 2020 8 February 2020

Please cite this article as: S. Yüksel, M. Pekcan, N. Puralı, G. Esendağlı, E. Tavukçuoğlu, V. Rivero-Arredondo, L. Ontiveros-Padilla, C. López-Macías, S. Şenel, Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan, International Journal of Pharmaceutics (2020), doi: https:// doi.org/10.1016/j.ijpharm.2020.119129

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Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan Selin Yüksel1, Mert Pekcan2, Nuhan Puralı3, Güneş Esendağlı4, Ece Tavukçuoğlu4, Vanessa Rivero-Arredondo5, Luis Ontiveros-Padilla5, Constantino López-Macías5,*, Sevda Şenel1*

1Faculty

of Pharmacy, Department of Pharmaceutical Technology, Hacettepe

University, 06100-Ankara, Turkey 2Faculty

of Veterinary Medicine, Department of Biochemistry, Ankara University,

Dışkapı, 06110- Ankara, Turkey 3Faculty

of Medicine, Department of Biophysics, Hacettepe University, 06100- Ankara,

Turkey 4Department 5Unidad

of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey

de Investigación Médica en Inmunoquímica, Hospital de Especialidades

Centro Médico Nacional ‘Siglo XXI’, Instituto Mexicano del Seguro Social (IMSS), Mexico City, Mexico * Corresponding Authors: [email protected]; [email protected] Keywords: chitosan; porins; adjuvant; vaccine; nanoparticles; microparticles

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ABSTRACT In order to improve the immunogenicity of the highly purified vaccine antigens, addition of an adjuvant to formulation, without affecting the safety of the vaccine, has been the key aim of the vaccine formulators. In recent years, adjuvants which are composed of a delivery system and immunopotentiators have been preferred to induce potent immune responses. In this study, we have combined Salmonella Typhi porins and chitosan to develop a new adjuvant system to enhance the immunogenicity of the highly purified antigens. Cationic gels, microparticle (1.69±0.01μm) and nanoparticles (337.7±1.7 nm) based on chitosan were prepared with high loading efficiency of porins. Cellular uptake was examined by confocal laser scanning microscopy, and the macrophage activation was investigated by measuring the surface marker as well as the cytokine release in vitro in J774A.1 macrophage murine cells. Porins alone were not taken up by the macrophage cells whereas in combination with chitosan a significant uptake was obtained. Porins-chitosan combination systems were found to induce CD80, CD86 and MHC-II expressions at different levels by different formulations depending on the particle size. Similarly, TNF-α and IL-6 levels were found to increase with porins-chitosan combination. Our results demonstrated that combination of porins with chitosan as a particulate system exerts enhanced adjuvant effect, suggesting a promising adjuvant system for subunit vaccines with combined immunostimulating activity.

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1. INTRODUCTION Immunization is based on the principal of generating an immune response to stimulate the immune system following administration of the disease-causing pathogen (antigen) to the body and to provide resistance against the disease-causing pathogen (Ogra et al., 2001). It has been a proven tool for controlling and eliminating life-threatening infectious diseases and is estimated to prevent between 2 and 3 million deaths each year . Hence, vaccination has been one of the most cost-effective health investments, with proven strategies that make it accessible to even the most hard-to-reach and vulnerable populations. Vaccines are broadly classified as live or inactivated. Live vaccines contain antigen that may be a weakened or killed form of the disease-causing organism, or fragments of the organism, whereas inactivated vaccines contain ‘wild’ viruses or bacteria that have been grown in a culture medium and inactivated before being included in a vaccine, or are made using a toxin, protein or polysaccharide (sugar) fragment derived from viruses or bacteria (subunit vaccines). Due to the safety issues, there has been a move towards the development of subunit vaccines that contain one or more defined microbial components instead of whole microorganisms. However, because that many of the pathogenic features of the microbe have been removed, these highly pure antigens exhibit lower immunogenicity, in contrast to live attenuated or inactivated whole-cell vaccines. Therefore, there has been a growing need for adjuvants to increase the immunogenicity of the subunit vaccines (Christensen, 2016; Del Giudice et al., 2018; Karch and Burkhard, 2016; Moyle, 2017; Moyle and Toth, 2013; O'Hagan et al., 2017).

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There are however only a few examples of adjuvants that have been used in licensed vaccines for human use, and there is still a need for new safe and effective adjuvant systems. In general, two approaches have been investigated as adjuvants: development of delivery systems, which help to improve the uptake and presentation of antigens, and development of immunopotentiators, which help to activate the innate immune system (Arca et al., 2009; O’Hagan et al., 2017; Schijns, 2017; Şenel, 2011). Combining immunostimulatory adjuvants with the delivery systems has been demonstrated to provide enhanced levels of response and/or focuses the response toward a desired pathway (Olafsdottir et al., 2015; Weinberger, 2018). The development of new adjuvants has been very slow due to the longer time needed to generate data to show the quality, efficacy and safety of the newly developed adjuvant. Especially safety of the adjuvants is very important. Aluminum, which has been used in vaccine formulations since 1930, is the only compound that has been approved as an adjuvant by itself (Hogenesch et al., 2018). All other adjuvants (eg., oil in water emulsions, liposomes, natural and synthetic toll like receptor ligands, virosomes) included in three marketed vaccines have approval with the product (Del Giudice et al., 2018; Garçon and Di Pasquale, 2017). The most common feature for the antigen delivery systems is their particulate nature. Particulates have comparable dimensions to the pathogens that are efficiently taken up by the antigen presenting cells (APC) of the immune system, and they offer further advantages such protection against antigen degradation, and acting as depot to prolong the residence time of the antigen (Lavelle and O'Hagan, 2006; McKee and Marrack, 2017; Shah et al., 2014). In addition to antigen transport, the particulate carrier systems themselves can increase the immune response.

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The composition as well as the particle size, surface charge and surface modification of the particulates play important role in inducing a certain type and strength of immune response (Bramwell and Perrie, 2006; Lebre et al., 2016; O'Hagan et al., 1991; Singh et al., 2007). Natural (e.g. chitosan, alginate, hyaluronic acid etc.) and synthetic polymers (e.g. polyanhydrides, polyesters (poly lactic acid, poly glycolic acid, poly (lactic-co-glycolic acid), poly caprolactone) and their combinations have been widely investigated for the preparation of polymeric particulates (Joshi et al., 2013; Sahdev et al., 2014; Yue and Ma, 2015). Chitosan is a cationic polymer obtained from the alkaline deacetylation of chitin, which is a glucose-based unbranched polysaccharide widely distributed in nature as the principal component of exoskeletons of crustaceans and insects as well as of cell walls of some bacteria and fungi (Arca et al., 2009; Illum et al., 2001; Scherließ et al., 2013; Şenel, 2019; van der Lubben et al., 2001). For its promising features such as

bioadhesivity, biocompatibility, biodegradability and penetration

enhancing activity, as well as immunostimulating activity, chitosan and its derivatives has been investigated both as an adjuvant/ antigen-delivery system, in different forms (gel, aqueous dispersion, micro and nanoparticle) for systemic and mucosal immunization of human and animals (Arca et al., 2009; Carroll et al., 2016; Çokçalışkan et al., 2014; Dhakal et al., 2018; Günbeyaz et al., 2010; Li et al., 2017; Li et al., 2018; Maxwell et al., 2006; Moran et al., 2018; Sayın et al., 2009; Sayın et al., 2008; Şenel, 2011; Şenel and McClure, 2004; Zaharoff et al., 2007). The studies being performed for over 30 years have shown that chitosan is able to enhance both humoral and cellular immune responses against various antigens. Yet, the mechanism of immunostimulating action has been elucidated only recently. The intracellular signaling pathways are reported to involve cGAS-STING, and NLRP3 (Carroll et al., 2016).

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Versatility in the physicochemical properties of chitosan can provide an excellent opportunity to engineer antigen-specific adjuvant/delivery systems. Several microbial components have also been used as vaccine adjuvants with their inherent properties to activate innate immune responses (Coffman et al., 2010; Garçon et al., 2011). Porins which are open channels filled with water in protein structure found in the outer membrane of gram-negative bacteria have been investigated in vitro and in vivo as vaccine adjuvants (Galdiero, 2003b; Hancock, 1987; Koebnik, 2002; Nikaido, 2003). Salmonella enterica serovar Typhi (S. Typhi) outer-membrane proteins (Omp) known as porins were investigated as a new typhoid vaccine in healthy volunteers, and strong specific antibody responses were found to be induced, and a cell-mediated immune response was obtained. Moreover, the porins-based vaccine did not induce significant adverse effects in vaccinated human volunteers (Blanco et al., 1993; Pelayo et al., 1989). Long-term protective IgM responses were shown to be elicited by S. Typhi outer-membrane protein C– and F–based subunit vaccine after 10 years (PerezShibayama et al., 2014). Lopez-Macias et al. (Cervantes-Barragan et al., 2009) have shown that S. Typhi porins represent not only a suitable B-cell antigen for vaccination, but also exhibit potent TLR-dependent stimulatory functions on B cells and DC, which help to further enhance and shape the antibody response. Further, OmpS1 and OmpS2 were shown to exert adjuvant effects in mice when co-immunized with the Vi capsular antigen from S. Typhi or inactivated 2009 pandemic influenza A(H1N1) virus [A(H1N1)pdm09] (Moreno-Eutimio et al., 2013). The adjuvant properties of S. Typhi porins, OmpC and OmpF against inactivated H1N1 2009 pandemic influenza virus were shown to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and Tindependent antigens in mice (Perez-Toledo et al., 2017).

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The aim of this study was to develop a new adjuvant/delivery system using the combination of the porins with the micro and nanoparticles prepared with the cationic polymer chitosan, which was used as an adjuvant and vaccine delivery system. Biocompatibility, cellular uptake, macrophage activation and cytokine release with the developed adjuvant systems was investigated in vitro in J774A.1 macrophage murine cells. 2. MATERIALS AND METHODS 2.1. Materials Water soluble chitosan, Protasan UP Cl 213 was purchased from NovaMatrix, Norway (Deacetylation degree: 75–90%; Mol.wt.: 150-400 kDa). Pierce™ BCA protein assay kit was purchased from Thermo Fisher Scientific (Waltham, MA USA). Propidium iodide (PI), Vybrant Dil (1,1 dioctadecyl, 3,3,3’,3’-tetramethyl indocarbocyanine perchlorate) Cell-Labelling solution, Alexa Fluor 488 conjugated Streptavidin and EZ Link NHS Biotin were purchased from Thermo Fisher Scientific (Waltham, MA USA). Tripolyphosphate (TPP), Bovine Serum Albumin (BSA), Accutase® solution and Lipopolysaccharides (LPS) from Escherichia coli O111:B4 were purchased from Sigma (Poole, UK). Anti-CD80-PE/Cy7 (16-10A1), CD86-PE (GL-1), I-Ad-Alexa647 (the mouse analogue of Class II Major histocompatibility complex-MHC); (39-10-8), and F4/80-FITC (BM8), LEGENDplex™ Mouse Th Cytokine Panel and propidium iodide dye were purchased from Biolegend (San Diego, USA). RPMI 1640 Medium with L-Glutamine and Certified Fetal Bovine Serum (FBS), Heat Inactivated were purchased from Biological Industries (Connecticut, USA). All other chemicals used were reagent grade.

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2.2. Porins production Porins were purified from S. Typhi ATCC 9993, as described previously (CervantesBarragan et al., 2009; Nikaido and Rosenberg, 1983; Salazar-González et al., 2004). Lipopolysaccharide (LPS) content was shown to be negative using the limulus amoebocyte lysate (LAL) assay (Charles River Endosafe Laboratories). 2.3. Preparation of Formulations Water soluble chitosan (Protasan UP Cl 213) was used for preparation of gel, microparticle and nanoparticle formulations. Chitosan gel at 0.5 % (w/v) concentration was prepared in distilled water. Porins were incorporated into chitosan gel at 0.02 % (w/v) concentration. Chitosan microparticles were prepared by spray drying using Buchi Mini Spray Dryer B 290 (Switzerland) (Günbeyaz et al., 2010). 0.1% (w/v) chitosan was dispersed in distilled water. 4 mL 1% (v/v) glutaraldehyde solution as crosslinker was added to 100 mL chitosan dispersion at the final concentration of 0.04 % (v/v) and stirred for 30 min on magnetic stirrer at room temperature. Spray drying was performed using a nozzle with diameter of 1.4 mm, the inlet temperature 1400C, outlet temperature 1100C, aspirator rate 85%, pump rate 5%. For porins loaded microparticles, porins were added to 0.1% (w/v) chitosan solution at a weight ratio of 1:100 (porins:chitosan). 4 mL glutaraldehyde solution at 1% (v/v) concentration was added to 100 mL chitosan+porins dispersion to have the final crosslinker concentration of 0.04 % and stirred on a magnetic stirrer for 30 min at room temperature. Spray drying was performed as described above. Chitosan nanoparticles were prepared by ionic gelation method (Sayın et al., 2008). 0.2% (w/v) chitosan was dispersed in distilled water. 0.1 % (w/v) TPP solution was used as crosslinker. Chitosan nanoparticles, with Chitosan/TPP ratio 5:1 were formed

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instantaneously by drop by drop addition of 2 mL 0.1 % (w/v) TPP solution to 5 mL 0.2% (w/v) chitosan solution,

stirring for 30 min on magnetic stirrer at room

temperature. Porins were loaded to nanoparticles, by adding % 0.1 (w/v) porins to 0.2 % (w/v) chitosan dispersion before adding the crosslinker, TPP. To remove unloaded porins, nanoparticle suspension was centrifuged at 15 000 rpm for 30 min.

2.4. Characterization of Formulations Viscosity of gels was measured using 2.4 mm diameter cone spindle (cone-CP52) on Cone Plate Viscometer (Model DV-II, Brookfield, Middleboro, USA) at 25 0C. Sample volume was 500 L. All measurements were performed in triplicate. Bioadhesion measurements were performed using Texture Analyzer (TA.XTPlus, Stable Micro Systems, Surrey, UK) equipped with 500 g load cell and removable aluminum probe. Synthetic membrane (MWCO: 12–14.000, Spectral Por, Thomas Scientific, NJ, USA), which was kept in water overnight before the experiment, was used to attach the formulations. The formulations were applied on the membrane and after contact time of 60 s, the probe was moved upward. Work of adhesion was calculated from the area under force versus time curve. The area under curve (AUC) was calculated from the force–time plot and calculated as the work of adhesion (mJ/cm2) using the equation given below (Eq. 1). Each experiment was performed in triplicate for each formulation. Work of adhesion (mJ cm2) = AUC / r2

(Eq 1)

The particle size of the microparticles was measured using Malvern Mastersizer 2000 (Malvern Instruments, UK).

Measurements were performed after suspending the

microparticles in isopropyl alcohol at 2 mg/mL concentration.

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The average particle size was expressed as the volume-weighted median diameter and the particle size distribution, in terms of SPAN factor. The particle size and polydispersity index (PDI) of the nanoparticles was measured using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). Nanoparticles were resuspended in distilled water for measurements. Surface charge of the formulations was determined using Zetasizer Nano Series, Nano-ZS (Malvern Instruments, UK). Measurements were performed in triplicate after resuspending the samples in distilled water. Nanoparticles were examined under transmission electron microscopy (FEI Tecnai G2 Spirit BioTwin CTEM, FEI Company, USA), and microparticles under scanning electron microscopy (QUANTA 400F Field Emission SEM, Thermo Fisher Scientific, USA). Loading efficiency (LE) of porins was calculated using Eq. 2. Porins were determined using the BCA protein assay.

LE= (Total amount of porins) - (Amount of unloaded porins) x 100 (Eq. 2) (Total amount of porins)

2.5. In Vitro Studies J774A.1 murine macrophage cell line (ATCC® TIB-67™), which is accepted as an appropriate in vitro model for functional studies such as phagocytosis, responsiveness to LPS, and proinflammatory cytokine production (Ralph and Nakoinz, 1977; Ralph et al., 1975), was cultured in RPMI 1640 (Biological Industries, Kibbutz Beit HaEmek, Israel) containing 20 % fetal bovine serum (FBS; Biological Industries), L-glutamine (2 mM), penicillin (100 U/mL) and streptomycin (100 μg/mL) at 37°C in a humidified 5% CO2 incubator.

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Release of porins The porins-loaded particles were dispersed in 2 mL phosphate buffer (pH 7.4) to obtain a 1% (w/v) suspension. Samples were incubated in water bath shaker (MEMMERT GmbH + Co. KG, Germany) and shaken at speed of 90 strokes per minute (spm) at 370C. Release studies were performed in triplicate. At appropriate time intervals, 250 μL samples were withdrawn and replaced with the same amount of phosphate buffer. The released amount of porins was determined using the BCA protein assay.

Biocompatibility studies The biocompatibility of the formulations containing the porins at different concentrations (2 g/mL, 1 g/mL, 0.5 g/mL, 0.25 g/mL) was determined as percent viability of J774A.1 cells using propidium iodide (PI) exclusion method. Following 24h incubation with the formulations (2x105 cells/400 µL culture medium in 48-well plate), the cells were harvested by a brief trypsin/EDTA treatment in phosphate buffer saline (PBS, pH 7.0) and stained with PI. The analyses were performed on a flow cytometer (FACSAria II, Becton Dickinson, San Jose, CA, USA)

Cellular Uptake High affinity biotin-avidin conjugation was used to examine the cellular uptake of the porins loaded formulations by a three-dimensional fluorescent microscopy method in J774A.1 cells. In this way, porins attached to biotin can be detected and isolated with fluorescently labelled avidin molecules. Porins were conjugated with biotin, and gel, micro and nanoparticle formulations were prepared in the same manner as described above, except for loading with biotinylated porins.

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The formulations were suspended in cell medium (RPMI 1640+ 20% FBS) (1:100 dilution) and inoculated on glass bottom dishes containing J774A.1 cells, which were stained with Vybrant Dil (1,1 dioctadecyl, 3,3,3’,3’-tetramethyl indocarbocyanine perchlorate) cell-labelling solution. The cells were incubated with formulations in a humidified incubator at 5% CO2 for 24 h. After 24 h incubation, cells were washed for three times with PBS. After fixation with 4% paraformaldehyde, the cells were permeabilised in 0.1% Triton X100 and blocked in 1 % BSA, respectively. The cells were washed and incubated in 0.1 g/mL Streptavidin-Alexa Fluor 488 solution to bind to biotin. The cellular uptake of the formulations was examined under confocal laser scanning microscope (Zeiss 200M fluorescent microscope equipped with a Laser scanner, LSM Pascal) with 40x objective oil immersion. 488 and 543nm laser lines from Argon and He–Ne Lasers were used for excitation of the Vybrant Dil and Alexa Fluor 488, respectively.

In vitro macrophage activation and cytokine release In vitro activation of macrophages was investigated by determining the expression of surface markers (CD80, CD86, MHC-II and F4/80) and release of cytokines. LPS was used as the positive control in the studies. J774A.1 macrophages were seeded onto 48-well plates (2x105 cells/well) and incubated with the formulations (1 g/mL in cell culture medium) for 24h. The cells cultured in medium alone were used as the negative control and LPS (1 µg/mL) stimulation was used as the positive control.

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At the end of incubation period, the cell culture supernatants were collected for cytokine analyses, and the cells were harvested by Accutase® treatment in PBS for the analysis of surface molecules. For evaluation of macrophage activation, the cells were labelled with monoclonal antibodies (mAb) anti-CD80-PE/Cy7 (16-10A1), CD86PE (GL-1), I-Ad-Alexa647 (the mouse analogue of Class II MHC; (39-10-8), and F4/80FITC (BM8). Following the removal of unbound mAb, immunophenotyping was performed by flow cytometry. The median fluorescence intensity (MFI) values for each surface molecule was determined. The cells incubated with isotype-matched antibodies served as technical controls. For cytokine release studies, the supernatants were analyzed by using a flow cytometry-based multiplex cytokine array (LEGENDplex™ Mouse Th Cytokine Panel (IL-5, IL-13, IL-2, IL-6, IL-9, IL-10, IFN-γ, TNF-α, IL-17A, IL-17F, IL-4, IL-21, IL-22), BioLegend) according to the manufacturer’s protocol. The data were collected using flow cytometry and cytokine levels were quantified with LEGENDplex™ data analysis software.

2.6. Statistical analysis All experiments were carried out at least in triplicate. Statistical analyses were performed using analysis of variance (ANOVA) test. When significant differences were indicated, the groups were further evaluated by multiple comparison tests.

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3. RESULTS 3.1. Characterization of Formulations Gels The viscosity of the chitosan gels (Chi Gel) was found to be 7.160.18 cP at shear rate: 200 s-1, showing Newtonian flow (Fig. 1) and viscosity was decreased in presence of porins (Porins+Chi Gel) (4.140.5 cP) at shear rate: 200 s-1 (p<0.001) (Fig.1). pH values for chitosan gel, porins+chitosan gel and porins were 5, 5.5 and 7, respectively.

Fig. 1. Flow properties of gel formulations (25  0.10C) (n=3) The bioadhesion work of the chitosan gel was found to be 0.55±0.050 mJ/cm2, whilst in presence of porins it was found to be 0.65±0.012 mJ / cm2 (p <0.1) (Fig 2).

Fig. 2. Bioadhesion properties of gel formulations (n=3) (* p < 0.1)

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Chitosan gel was found to have positive zeta potential (78,1±2,0), whilst porins alone showed negative zeta potential (-19.2±1.9).

When porins were incorporated into

chitosan gel, the zeta potential was found to be positive (36.3±0,71).

Microparticles Spherical microparticles of 1.3-1.7 m particle size were obtained (Fig. 3, Table 1). Loading efficiency was found to be above 70%. Particle size was found to increase with porins loading. Zeta potential of the microparticles was positive, and a decrease in zeta potential was observed with porins loading (Table 1).

Fig. 3. SEM micrographs of chitosan microparticles: blank (A) and porins loaded (B)

Nanoparticles Positively charged nanoparticles with a narrow particle size distribution were obtained (Table 1, Fig. 4), with high porins loading (>85 %). No change in particle size was observed with loading of porins. The positive zeta potential of the nanoparticles was found to decrease in the presence of porins.

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Fig. 4. TEM micrographs of chitosan nanoparticles: blank (A) and porins loaded (B)

Table 1. Properties of particle formulations Formulation

Code

Particle Size ± S.D.

SPAN± S.D.

Chitosan blank microparticle

Chi MicroP

1.30±0.01 mm

3.18±0.02

Porins loaded microparticle

Porins+ Chi MicroP

1.69±0.01 mm

Chitosan blank nanoparticle

Chi NanoP

Porins loaded nanoparticle

Porins+ Chi NanoP

PDI± S.D.

Zeta potential (mV) ± S.D.

Loading Efficiency (%)± S.D.

-

33.5±1.60

-

1.00±0.03

-

32.56±0.78

791.3

366.4±4.8 nm

-

0.20±0.01

47.9±3.5

-

337.7±1.7 nm

-

0.29±0.02

30.3±0.5

86.5±4.70

*S.D.: standard deviation

3.2. In vitro studies 3.2.1. Release of porins Release of porins was found to be below 20% and very slow during the study period indicating the in vitro stability of the particles (Fig. 5).

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Fig. 5. In vitro release of porins from microparticles and nanoparticles (n=3)

3.2.2. Biocompatibility The viability of J774A.1 murine macrophage cells was found to be above 80% when porins and chitosan were administered alone (Fig. 6). Similarly, the cell viability was not reduced with the porins-loaded gel and nanoparticle formulations, whilst there was a slight increase in the cell death (viable cells, 60-70%) with the blank microparticles and the porins-loaded microparticles.

Fig. 6. % viability of J774A.1 cells following administration of the formulations (n=3)

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3.2.3. Cellular Uptake Porins were observed to be accumulated around the cell membrane when they were applied alone, without being taken into cells (Fig. 7b), whereas in presence of chitosan (Porins+Chi Gel), cellular uptake of porins was obtained (Figs. 7c, 8a).

With

nanoparticles, formation of aggregates was observed, which possibly reduced the cellular uptake (Fig. 7d). The highest cellular uptake was observed with microparticle formulations (Figs 7e, Fig 8b).

Fig. 7. Cellular uptake of formulations by J774A.1 cells. Biotinylated porins were conjugated with Alexa Fluor 488- Streptavidin and the cells were stained with Vybrant Dil (red). A) untreated cells (control); B) Porins; C) Porins+Chi Gel; D) Porins+Chi NanoP; E) Porins+Chi MicroP

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Fig. 8. Three dimensional confocal images of uptake of Porins+Chi gel (A) and Porins+Chi MicroP (B) by J774A.1 murine macrophage cells (biotinylated porins were conjugated with Alexa Fluor 488- Streptavidin and the cells were stained with Vybrant Dil (red) (a) cross-section of projection image in z, x plane; (b) z-stack of images in xy plane (focal section: 5/17, section thickness: 0.367 m); (c) cross-section of projection image in z, y plane).

3.2.4. Macrophage Activation and Cytokine Release When given alone, porins, chitosan microparticles and nanoparticles induced expression of CD80 at similar levels with that of the positive control (LPS) (p> 0.1) whereas no significant stimulation was found with chitosan gel (Fig.9a). Expression of CD80 molecule was found to increase significantly when porins and chitosan were combined. The highest expression of CD80 was obtained with the porins incorporated into chitosan gel (Porins+Chi Gel) (p <0.001). Porins and chitosan formulations induced higher levels of CD86 expression than the positive control (Fig. 9b). Combination of porins with chitosan in gel form resulted in higher CD86 expression when compared to that with porins and chitosan gel alone (p <0.001).

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With the porins loaded nanoparticles, a slight increase in CD86 level was observed when compared to porins alone, whilst no change was observed with the microparticle formulations. No significant MHC-II expression was observed with the porins alone (Fig. 9c). A significant increase was obtained in MHC-II expression when combined with chitosan (p <0.1). Similar increase was observed for gel and nanoparticle formulations, whilst with microparticle formulations the MHC-II expression was the highest (p <0.01). Porins were observed to upregulate the expression of F4 / 80 yet not reaching to that of the cells stimulated with LPS, which was used as positive control (p> 0.1) (Fig. 9d). Chitosan was found to stimulate the expression of F4/80. When the porins were combined with chitosan, an increase in F4/80 expression was obtained. B 2500

A 12000 *

***

****

****

*

**

***

6000

Control (-) Control (+) Porins Chi Gel Porins+Chi Gel Chi NanoP Porins+Chi NanoP Chi MicroP Porins+Chi MicroP

****

2000 ***

4000

*

****

**

* ****

***

Control (-) Control (+) 1500 Porins Chi Gel Porins+Chi Gel Porins+Chi Chi NanoPNanoP 1000

*

**

* ***

Chi MicroP Porins+Chi MicroP 500

M

ic ro P

oP

ic ro P M

hi s+ C

C

rin Po

hi s+ C

rin

F4/80 MFI

Po

hi

N

N hi C

rin s Po

an

G el

an

G el

hi +C

rin s

*

hi

l( +) tro

on

Control800 (-) Control (+) Porins Chi Gel 600 Porins+Chi Gel Chi NanoP Porins+Chi NanoP 400 Chi MicroP

C

* 1000

on

tro

*

oP

Formulations 1000

l( -)

D

Po

Formulations 1500

C

2000

500

l( +)

200

hi G el Ch iN an oP ri n s+ C hi N an oP C hi Po Mi ri n cro s+ P C hi Mi cro P

Formulations

G el Ch iN an oP s+ C hi N an oP C h iM Po ic ri n ro s+ P C hi Mi cro P ri n

Po

Po

ri n s+ C

hi

s Ch iG el

(+)

ri n

ro l

Po

l( -) on t C

C on tro

Po

Po

ri n

s+ C

Ch iG el

s

(+)

ri n

ro l

Po

on t C

on tro

l( -)

Formulations

C

Control (-) Control (+) Porins Chi Gel Porins+Chi Gel Chi NanoP Porins+Chi NanoP Chi MicroP Porins+Chi MicroP

Po

tr o

on tr o

C on

C

rin s Ch iG Po e r in l s+ C hi G el Ch iN Po an r in oP s+ C hi N an oP C hi Po Mi r in cr s+ oP C hi Mi cr oP

Porins+Chi MicroP

l( -)

MHC-II MFI

**** **

C

CD80 MFI

8000

C

*

CD86 MFI

10000

***

** ****

Fig. 9. Expression of surface markers by J774A.1 cells at 24 h after application of the formulations: A) CD80; B) CD86; C) MHC-II; D) F4/80. Negative control: untreated cells: positive control: LPS-treated cells (*p<0.1, **p<0.01, ***p<0.001, ****p<0.0001) (n=3) 20

When the cytokine release was examined, TNF-α and IL-6 were found to be the only cytokines secreted by the J774.A1 cells upon application of the formulations (Fig.10). No significant change was found in the released amounts of other cytokines (IL-5, IL13, IL-2, IL-6, IL-9, IL-10, IFN-γ, TNF-α, IL-17A, IL-17F, IL-4, IL-21, IL-22) when compared to the control (data not shown). When given alone porins, chitosan gel and chitosan nanoparticles were observed to have no effect on TNF-α release, except for chitosan microparticles (Fig. 10a). When porins were combined with chitosan in gel and microparticle form, TNF-α levels were found to increase significantly, the highest TNF-α level with the microparticles (Porins+Chi MicroP) (p<0.001). With porins loaded nanoparticles (Porins+ Chi NanoP), no significant TNF-α release was observed. When given alone, porins, chitosan gel, chitosan micro and nanoparticles were observed to have no effect on IL-6 release (Fig. 10b). Combination of the porins with chitosan gel, a significant increase in release of IL-6 was obtained (p <0.0001), whilst with micro and nanoparticles no increase was observed when compared to positive control.

150

Porins

Chi Gel B

Porins+Chi Gel

*

***

Chi NanoP

Porins+Chi NanoP

Chi MicroP Porins+Chi MicroP

400

*** **

*

*

300

**

**

Control (-) 200 Control (+) Porins Chi Gel 100 Porins+Chi Gel Chi NanoP Porins+Chi NanoP 25 Chi MicroP 20 Porins+Chi MicroP 15 10 5 0

100

IL-6 pg/mL

50

Formulations

s

ri n s+ C

Po

Po

ri n

(+)

Ch iG el

Po

(-)

tro l

ro l

on C

on t C

Po

ri n

s+ C

hi G el Ch iN Po an ri n oP s+ C hi N an oP C hi Po M i ri n cro s+ P C hi Mi cro P

s ri n

Ch iG el

Po

l( +) tro

on C

C on

tro

l( -)

Formulations

hi G el Ch iN an oP ri n s+ C hi N an oP C hi Po Mi ri n cro s+ P C hi Mi cro P

TNF- pg/mL

Control (+)

Control (-)

A

Fig. 10. Cytokine release by J774.A1 cells at 24 h after application of the formulations: A) TNF-α and B) IL-6 . Negative control: untreated cells: positive control: LPS-treated cells (*p<0.1, **p<0.01, ***p<0.001) (n=3).

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4. DISCUSSION Highly purified subunit vaccines contain only the antigenic parts of the pathogen and by this means they provide higher safety, however due to the removal of pathogenic features of the organism, they are often less immunogenic (Di Pasquale et al., 2015; Moyle, 2017). In order to enhance the immune responses, they need to be combined with adjuvants, which are capable of enhancing and/or modulating immune responses by exposing antigens to APCs. The particulate delivery systems help to improve the uptake and presentation of antigens due to their similar size and structure as that of a pathogen, and the immunopotentiators help to activate the innate immune system (O’Hagan and Singh, 2003; Oyewumi et al., 2010; Shah et al., 2014; Xiang et al., 2006; Yue and Ma, 2015). With the combination of both approaches provides the best opportunity to produce highly potent vaccines. Previously, we have shown in vitro and in vivo that chitosan microparticles and nanoparticles are promising adjuvant/delivery systems for several experimental antigens (Çokçalışkan et al., 2014; Günbeyaz et al., 2010; Sayın et al., 2008; Şenel, 2011). On the other side, Lopez-Macias and his team have shown that S. Typhi outer membrane proteins (porins) are versatile vaccine adjuvants, which could be used to enhance T-cell immune responses toward a Th1/Th17 profile, while improving antibody responses to otherwise poorly immunogenic T-dependent and T-independent antigens (Moreno-Eutimio et al., 2013; Perez-Toledo et al., 2017). Based on the gained experiences, we aimed to develop a particulate adjuvant system which is composed of chitosan and porins. We combined porins and chitosan in micro- and nanoparticle forms. For comparison, porins were also incorporated into chitosan gel at a suitable viscosity for administration.

22

Chitosan microparticles (1.3 to 1.7±0.01m) with high porins loading efficiency (> 80%) were prepared using spray drying method, which is a rapid, reproducible and easily scalable process. Both the blank and the porins loaded microparticles had positive surface charge (Table 1). Nanoparticles were also prepared with high loading porins efficacy (> 85 %) and with positive surface charge and narrow size distribution (330 to 370 nm) (Table 1). The integrity of the porins during preparation of both micro- and nanoparticles was confirmed with SDS page (data not shown). Furthermore, very low release of porins was obtained, which indicated the in vitro protection of porins. Cellular uptake of the formulations was found to be dependent on the surface charge of the formulations. Porins alone, which has negative surface charge were not able to enter the cells (Fig 7b) whereas, cellular uptake was obtained when combined with chitosan, because the surface charge shifted to positive. correlation with the previous studies reporting

These results

shows

that cellular uptake of cationic

formulations are higher due to the interaction with the negatively charged cell, which plays crucial role in the interaction between particles and APCs (Foged et al., 2005; Lerch et al., 2013; Shah et al., 2014). The combined adjuvant systems developed in this study would provide enhanced immune response due to their positive surface charge. Viability of the cells was observed to be higher with gels and microparticles (>80 %), whilst a decrease was observed with microparticles (60-70%). This can be attributed to particle size difference, where microparticles with larger particle size than that of the nanoparticles affecting the macrophages more strongly (Foged et al., 2005).

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On the other hand, cellular uptake of the microparticles were found to be higher when compared to that of nanoparticles, which aggregated and formed a non-spherical cluster. It has been reported that spherical particles were more readily taken up when compared to non-spherical particles (Kumar et al., 2015; Mathaes et al., 2015). This would be one of the reasons for higher uptake of our microparticles. In general, particles which have a particle size similar to that of a pathogen (<10 µm) are effectively taken into the cell and induce the immune response. Especially, particles smaller than 5 µm are taken by phagocytosis and are recognized by macrophages (Bachmann and Jennings, 2010; Cruz et al., 2010; Foged et al., 2005; Kumar et al., 2015; Li et al., 2011; Mathaes et al., 2015; O’Hagan and Singh, 2003; Oyewumi et al., 2010; Silva et al., 2015; Xiang et al., 2006). Lerch et al (Lerch et al., 2013) have reported that the uptake of the particles depends not on the number of particles but on the total amount of polymeric material present in the media (Lerch et al., 2013). They demonstrated that the total surface area of the particle size did not to correlate linearly with the uptake, and they concluded that there was no direct dependency between the total surface area and the cellular endocytic process to overcome the cell membrane.

This

interpretation can be another explanation for our results which showed higher cellular uptake with microparticles from the macrophage cells. Furthermore, in another study, it was demonstrated that the microparticles (2 m) were taken up nonspecifically and the nanoparticles effectively targeted the human dendritic cells (Cruz et al., 2010). Particles of 20−200 nm have been reported to efficiently enter the lymphatic system, whereas particles that are larger than 200−500 nm did not enter lymph capillaries in a free form and they needed to be carried into the lymphatic system by specialized cells, such as dendritic cells or macrophages (Bachmann and Jennings, 2010).

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Using nanoparticles to deliver antigens, the efficiency of uptake into dendritic cells was shown to significantly increase compared to soluble antigen alone (Gregory et al., 2013). Smaller-sized nanoparticles (<40 nm) were shown to be

taken up more

efficiently, but larger-sized nanoparticles (>200 nm) could delivered a greater amount of antigen to APCs (Shima et al., 2013) . Our results showed that the both micro- and nanoparticles are taken up in vitro by the macrophage cells, higher uptake with the microparticles. These results can provide a perspective for in vivo studies, yet it must be noted that the results of the in vivo studies would also be dependent on the antigen used as well as the animal model. T-cells which are involved in cellular immune response need to be activated in order to differentiate into effector cells and to perform their functions. Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific Tcell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of APCs such as macrophages and dendritic cells. The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell and cytokine production (Abbas et al., 2007; Iwasaki and Medzhitov, 2010; Medzhitov, 2002; Vasilevko et al., 2002).

In our study, we investigated in vitro

modulation of surface molecule expression and cytokine secretion in order to demonstrate the ability of the developed adjuvant systems to induce macrophages activation. With combination of porins and chitosan in microparticle form (1-2 μm), significantly high expression of MHC-II was observed, whereas with nanoparticles (330 nm) MHC-II expression was not observed. This can be attributed to the difference in particle size, as it has been reported previously that uptake of chitosan particles by dendritic cells and macrophages was found to be dependent on particle size, higher

25

uptake with microparticles (1 μm) when compared to nanoparticles (300 nm. Similarly, Brewer et al (Brewer et al., 2004) have also reported that particle size modulated the efficiency of antigen presentation by murine macrophages. In another study, it was stated that microparticles (2-3 μm ) were effectively taken up by macrophages through receptor-mediated endocytosis and phagocytosis, whilst nanoparticles with their size allowing micropinocytosis, uptake of nanoparticles was favored more by dentritic cells when compared to the microparticles (Slütter and Jiskoot, 2016). Uptake of chitosan particles were found to enhance upregulation of surface activation markers on APCs and increased the release of pro-inflammatory cytokines. Our results showed that with porins alone or chitosan alone, expression of MHC-II molecules in murine macrophage cells was not observed. This is in correlation with the previous results reporting that the outer membrane proteins obtained from S. Typhi induced MHC II molecules in dendritic cells but not in macrophages (Moreno-Eutimio et al., 2013). There are other studies demonstrating that there can be differences in some of the responses between macrophages and dendritic cells (Kalupahana et al., 2005). For the combined adjuvant system, we can suggest that as a result of strong stimulation of MHC II expression, the microparticles would provide higher activation of T helper cells. CD80 and CD86 are the costimulatory molecules on the surface of APCs which play role in activation of the T cells.

Porins used in our study induced CD80 and CD86

expressions similar to that with LPS, which was used as the control, which is in correlation with the results reported on S. Typhi porins acting as be TLR agonists, hence inducing expression of CD80 and CD86 (Cervantes-Barragan et al., 2009; Chow et al., 1999; Galdiero, 2003a; Galdiero, 2003b). When porins were combined with chitosan in gel and nanoparticle form, CD80 and CD86 expression was observed

26

to increase, which was also in correlation with our in vitro cellular uptake results showing higher uptake of gels and nanoparticles. Similarly, Ed Lavelle and his team (Moran et al., 2018) have demonstrated that chitosan in solution induced expression of the CD86 costimulatory molecule on the surface of APCs in cellular immunity. On the other side, no significant increase in CD80 and CD86 expression was observed with the microparticle formulations, although the MHC II expression was highest with the microparticles. Furthermore, TNF- and IL-6 release was also found to be increased with chitosan+porins microparticles whilst no release was observed with the nanoparticles. Taking into consideration also the decreased cell viability with the microparticles, overall results indicate the activation of the macrophages by the developed combined adjuvant systems, upregulating the expression of surface proteins, MHC II, CD80 and CD86, which depends on the particle size of the systems. Furthermore, induction of TNF- and IL-6, which are the proinflammatory cytokines that induce APC maturation, in response to the developed adjuvant systems would facilitate the APC activation and initiate an adaptive immune response.

The F4/80

molecule is regarded as one of the most specific cell-surface markers for murine macrophages and it is involved in direct cell–cell contact between macrophages and NK cells that results in cell activation and optimal cytokine production (Lin et al., 2005). Significant expression of F4/80 was observed with the chitosan+porins combined systems in gel and nanoparticle form. No significant release was observed for the cytokines tested (IL-5, IL-10, IL-13, IL-2, IL-9, IFN-γ, IL-17A, IL-17F, IL-4, IL-21, IL-22), other than TNF- and IL-6.

Absence of these cytokines’ release can indicate

distinctive immune responses. For example, IL-10, which is produced by macrophages and T cells (especially regulatory T cells), provides inhibition of antigen presentation (Abbas et al., 2007; Saraiva and O'Garra, 2010) and its absence has been reported to

27

resulted in enhanced antigen-specific Th1 responses after vaccination. Oleszka et al (Oleszycka et al., 2018) have reported that injection of alum promoted IL-10, which can block Th1 responses and may explain the poor efficacy of alum as an adjuvant for inducing protective Th1 immunity. Hence, with our chitosan+porins based systems, absence of IL-10 release indicates enhanced Th1 responses.

Properties of the

chitosan such as molecular weight, deacetylation degree and pattern as well as the amount of chitosan used in the adjuvant systems, the particle number and total interaction surface would also have an effect on these responses (Fong and Hoemann, 2018; Lerch et al., 2013). Hence, it must be kept in mind that when a comparison is made with the results reported in the literature, due to the above mentioned variables and experimental designs, varying responses to chitosan can be observed.

5. CONCLUSION In this study, we have developed a particulate adjuvant system composed of chitosan and porins that combines the immunostimulating effects of both components and can also serve as an antigen delivery system. Enhanced activation of immune system is aimed by these combined mechanisms. Microparticles and nanoparticles were obtained with positive surface charge, high loading efficiency, good in vitro stability and appropriate particle size as a vaccine adjuvant. The combined adjuvant systems were taken up by macrophage cells without showing any significant cell toxicity. Our results demonstrated that the newly developed combined particulate adjuvant systems act on macrophages, inducing the up-regulation of cytokines, MHC class II and costimulatory molecules, hence would lead to activation of the innate immune system and consequently increase the magnitude of the adaptive immune response.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CL-M is listed as inventor on a patent related to the use of Salmonella porins as adjuvants and vaccines. All remaining authors declare no competing interests.

Acknowledgements This project was performed through the bilateral collaboration supported by TÜBİTAKTurkey (SBAG- 215S995) awarded to SŞ and CONACYT-Mexico Project: SRECONACYT 263683 awarded to CL-M and the Mexican National Research Council (CONACYT-Mexico) Projects: SRE-CONACYT 263683 and SEP-CONACYT CB2015256402, awarded to CL-M.

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Figure Legends Figure. 1. Flow properties of gel formulations (25  0.10C) (n=3) Figure. 2. Bioadhesion properties of gel formulations (n=3) (* p < 0.1) Figure. 3. SEM micrographs of chitosan microparticles: blank (A) and porins loaded (B) Figure. 4. TEM micrographs of chitosan nanoparticles: blank (A) and porins loaded(B) Figure. 5. In vitro release of porins from microparticles and nanoparticles (n=3) Figure. 6. % viability of J774A.1 cells following administration of the formulations (n=3) Figure. 7. Cellular uptake of formulations by J774A.1 cells. Biotinylated porins were conjugated with Alexa Fluor 488- Streptavidin and the cells were stained with Vybrant Dil (red). A) untreated cells (control); B) Porins; C) Porins+Chi Gel; D) Porins+Chi NanoP; E) Porins+Chi MicroP Figure. 8. Three dimensional confocal images of uptake of Porins+Chi gel (A) and Porins+Chi MicroP (B) by J774A.1 murine macrophage cells (biotinylated porins were conjugated with Alexa Fluor 488- Streptavidin and the cells were stained with Vybrant Dil (red) (a) cross-section of projection image in z, x plane; (b) z-stack of images in xy plane (focal section: 5/17, section thickness: 0.367 m); (c) cross-section of projection image in z, y plane) Fig. 9. Expression of surface markers by J774A.1 cells at 24 h after application of the formulations: A) CD80; B) CD86; C) MHC-II; D) F4/80. Negative control: untreated cells: positive control: LPS-treated cells (*p<0.1, **p<0.01, ***p<0.001, ****p<0.0001) (n=3)

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Fig. 10. Cytokine release by J774.A1 cells at 24 h after application of the formulations: A) TNF-α and B) IL-6 . Negative control: untreated cells: positive control: LPS-treated cells (*p<0.1, **p<0.01, ***p<0.001) (n=3) Selin Yüksel: experimental, writing; Mert Pekcan: experimental; Nuhan Puralı: visualization; Güneş Esendağlı: cytokine studies; Ece Tavukçuoğlu: cytokine studies; Vanessa Rivero-Arredondo: porins preparation ;Luis Ontiveros-Padilla: porins preparation; Constantino López-Macías: porins preparation, conceptualization methodology, editing ; Sevda Şenel: methodology; supervision, writing- reviewing, conceptualization

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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