Protamine Nanocapsules for the Development of Thermostable

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Protamine Nanocapsules for the Development of Thermostable Adjuvanted Nanovaccines José Vicente Gonzalez-Aramundiz, Mercedes Peleteiro Olmedo, Africa González-Fernández, Maria Jose Alonso Fernandez, and Noemi Stefania Csaba Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00852 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on October 31, 2018

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Molecular Pharmaceutics

Protamine Nanocapsules for the Development of Thermostable Adjuvanted Nanovaccines José Vicente González-Aramundiz†‡, Mercedes Peleteiro Olmedo§, África González-Fernández§, María José Alonso Fernández†*, Noemi Stefánia Csaba†* †: Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Dept. of Pharmacology, Pharmacy and Pharmaceutical Technology, School of Pharmacy, Univ. of Santiago de Compostela, Santiago de Compostela, Spain. (E-mail address: [email protected], [email protected], [email protected]). §: Immunology, Centro de Investigaciones Biomédicas (CINBIO) (Centro Singular de Investigación de Galicia), Instituto de investigación sanitaria Galicia-Sur (IISGS), University Campus, Universidade de Vigo, Vigo, Spain (E-mail address: [email protected], [email protected]) ‡: Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago, Chile. (E-mail address: [email protected]) Corresponding Author * Prof. María José Alonso, E-mail address: [email protected]. Noemi Csaba, E-mail address: [email protected]. Tel: +34 881815454 / +34 981594488 ext. 15454. Full postal address:

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Center for Research in Molecular Medicine and Chronic Diseases – CIMUS. Av. Barcelona s/n, Campus Vida University of Santiago de Compostela. 15782 Santiago de Compostela, Spain.

TABLE OF CONTENTS GRAPHIC

ABSTRACT

One of the main challenges in the development of vaccine has been to improve their stability at room temperature and eliminate the limitations associated to the cold chain storage. In this paper, we describe the development and optimization of thermostable nanocarriers consisting of an oily core with immunostimulating activity, containing squalene or α tocopherol surrounded by a protamine shell. The results showed that these nanocapsules can efficiently associate the recombinant hepatitis B surface antigen (rHBsAg) without compromising its antigenicity. Furthermore, the freeze-dried protamine nanocapsules were able to preserve the integrity and bioactivity of the associated antigen upon storage for at least 12 months at room temperature. In vitro studies evidenced the high internalization of the nanocapsules by immunocompetent cells, followed by cytokine secretion and complement activation. In vivo studies showed the capacity of rHBsAg-loaded nanocapsules to elicit protective levels upon intramuscular or intranasal

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administration to mice. Overall, our data indicates that protamine nanocapsules are an innovative thermostable nanovaccine platform for improved antigen delivery.

KEYWORDS: nanocapsules, antigen delivery, protamine, thermostable, nanovaccine, hepatitis B.

ABBREVIATIONS: APC: antigen presenting cells CVF: cobra venom factor NC: Nanocapsules PdI: polydispersity index PRRs: pattern-recognition receptors rHBsAg: recombinant Hepatitis B surface antigen SQL: Squalene TCPH: α tocopherol Wb: Western blot assay

INTRODUCTION Polymeric nanocarriers have raised expectations in the field of antigen delivery due to their capacity to combine adjuvant properties with specific abilities for overcoming biological barriers. In fact, this behavior has rendered such nanocarriers a promising strategy towards achieving single

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dose and needle-free vaccines.1 Among the materials used in the development of antigen delivery, biodegradable polyesters, polysaccharides and polypeptides are probably the ones receiving more attention.2, 3 This is mainly due to their adequate safety profile and their promising performance during both preclinical research studies and clinical trials. More recently, there has been an increased interest on the use of biomaterials with inherent capacity to interact with the immune system, i.e. materials that are able to activate the pattern-recognition receptors (PRRs) of immunocompetent cells and modulate their cytokine secretion profile.4 For the purpose to create versatile drug delivery vehicles, our group has designed protamine nanocapsules (NC), and studied their potential for oral peptide delivery 5, ocular wound healing 6, and more recently, for immunization when carrying a protein antigen.7 We chose protamine as principal biomaterial for the elaboration of these NC because of its FDA approved status as a pharmaceutical excipient (NPH insulin) and also as a drug (heparin antagonist in cardiac surgery).8 In addition, protamine offers other interesting properties in the field of immunization, such as its cell penetrating capacity

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and the ability to enhance the response to specific antigens when

presented in the form of microparticles or nanoparticles.10-12 Based on this background information, our goal in this study, was to further explore the potential of protamine-based NC for immunization, and specifically expand our understanding of their mechanism of action, and their potential as a thermostable vaccine formulation. In order to rationally design and optimize these NC, we have selected two different types of oils as core materials: squalene and α tocopherol (vitamin E), both of them with proven adjuvant activity.13 In fact, squalene and α tocopherol have been included as adjuvants in marketed influenza vaccine formulations such as MF59 and ASO3.14

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Finally, to assess the potential of protamine NC for immunization we chose the recombinant Hepatitis B surface antigen (rHBsAg). Although this antigen gives high responses upon intramuscular injection when adjuvanted with alum15, significant problems remain to be solved in order to enhance its efficacy. For example, the alum-adsorbed vaccine needs to be stored in a narrow range of temperature (2-8 ºC), a fact that poses an important limitation on the distribution of this vaccine in developing countries.16 Moreover, this vaccine needs to be administered according to a multiple injection protocol. Our approach offers the possibility of a needle-free administration, which would be a major milestone towards improving immunization coverage worldwide. Based on these premises, in this paper we report the development and the comprehensive analysis of protamine NC as antigen delivery vehicles. Namely, the resulting nanovaccines were characterized according to their physico-chemical properties, their capacity to load and release rHBsAg, their stability upon storage as a freeze-dried powder, their ability to stimulate cytokine production in blood mononuclear cells, and their in vivo performance, following either intramuscular or intranasal administration. Our goal is to provide an overview of the potential of protamine NC in the development of advanced, thermostable, and safer vaccines.

EXPERIMENTAL SECTION Materials Protamine sulfate was purchased from Yuki Gosei Kogyo, Ltd. (Japan). Recombinant hepatitis B surface antigen (rHBsAg) was kindly donated by Shantha Biotechnics Limited (Hyderabad, India). Polyethylene glycol (PEG)-Stearate (Simulsol M52) was from Seppic (France). Squalene (SQL) and α tocopherol (TCPH) were supplied by Merck (Germany). Enzyme linked

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immunosorbent assay (ELISA) kit (Murex rHBsAg Version 3) was from Diasorin (United Kingdom). Antibodies for Western blot detection, chicken polyclonal antibody to hepatitis B virus surface antigen and rabbit polyclonal antibody to chicken conjugated with horseradish peroxidase were purchased from Abcam pcl (United Kingdom). For complement analysis by Western blot, mouse monoclonal antibody (mAb) against human complement factor 3 (C3) was obtained from Abcam (Cambridge, UK). Secondary polyclonal goat antibodies anti-mouse IgG conjugated to alkaline phosphatase were from Dako (Glostrup, Denmark). For ELISA, mouse monoclonal IgG and rabbit IgG against HBsAg were purchased from Acris Antibodies GmbH (Hiddenhausen, Germany) and Biokit (Barcelona, Spain), respectively. Secondary Abs (both polyclonal goat antibodies anti-rabbit and anti-mouse IgG, IgG1, IgG2a conjugated to horseradish peroxidase) were from Southern Biotech (Birmingham, AL). 5-Carboxytetramethylrhodamine succinimidyl ester (TAMRA) was from Invitrogen (United Kingdom). Fetal bovine serum (FBS), glutamine and penicillin/streptomycin were purchased by PAA (Austria). Glucose, trehalose, PBS, sodium cholate and aluminum hydroxide gel were obtained from Sigma-Aldrich (Spain). All other chemicals used were of reagent grade or higher purity.

Preparation of protamine nanocapsules Blank protamine nanocapsules (NC) were prepared by a solvent displacement technique. Briefly, PEG-stearate (12 mg) and sodium cholate (5 mg) were dissolved in ethanol (750 µl) and mixed with a solution of an oil, either TCPH (60 mg) or SQL (62.5 µl) in acetone (4.25 ml). The organic phase was immediately poured into 10 ml of aqueous phase with protamine (0.05 % w/v). The formation of the systems was evident due to the milky appearance of the mixture. The organic

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solvents were eliminated under vacuum (Heidolph, Germany), and NC were collected as an aqueous suspension with a constant volume of 5 ml. The isolation of the protamine NC either with SQL or with TCPH core was carried out by ultracentrifugation (Optima L-90K, Ultracentrifuge, Beckman Coulter; USA) at 61740 x g for 1h, at 15 ºC. The isolated NC were resuspended in ultra-pure water to a final theoretical protamine concentration of 1 mg/mL.

Characterization of protamine nanocapsules The hydrodynamic diameter and polydispersity index of the nanosystems were determined by Dynamic Light Scattering (DLS) (Zetasizer, NanoZS, Malvern Instruments, Malvern, UK), after sample dilution with ultrapure water. The zeta potential was measured by laser-Doppler anemometry by diluting the samples with 1 mM KCl. Morphological examination was carried out by transmission electron microscopy (TEM, CM12 Philips, Netherlands). The samples were stained with 2% (w/v) phosphotungstic acid solution.

Association of rHBsAg to protamine nanocapsules and determination of its structural integrity To prepare NC loaded with rHBsAg, equal volumes of the isolated protamine NC and the model antigen were incubated for 1h at room temperature, at a mass ratio of 4:1 (protamine:rHBsAg)17. The ability of the protamine NC to associate the rHBsAg antigen was determined by an indirect method: calculating the difference between the total amount of antigen used in the incubation process and the amount of free rHBsAg remaining in the aqueous medium after the isolation

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process. This amount of non-associated antigen was quantified by ELISA following the manufacturer’s instructions. Western blot assay (Wb) was used as a qualitative tool for the determination of the antigen’s structural integrity. For this purpose, we followed the procedure described in

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with minor

modifications. In our case, the primary antibody was incubated for 1 h at room temperature and the antibody–antigen complexes were visualized using the ECL Plus Western Blotting Detection Reagents (Amersham Biosciences, UK) in UVP imaging system (EC3™ Imaging System, UVP, USA).

In vitro release studies of rHBsAg from protamine nanocapsules In vitro release studies of the rHBsAg from protamine NC were performed to assess the ability of this system to release the associated antigen in its active form. Thus, 50 µl of isolated rHBsAgloaded protamine NC (TCPH core) were incubated with 1.95 ml of PBS (pH 7.4) with Tween 80 (0.02 % v/v) at 37ºC, under moderate shaking. Previously, we confirmed that the protamine NC remained stable under these conditions. At different time points (1, 4, 8 and 24 h), the suspension was ultracentrifuged, (as described above) and the remaining aqueous medium was analyzed by ELISA to quantify the released antigen. These samples were also analyzed by Western blot in order to check the structural integrity of the antigen.

Stability of rHBAg-loaded freeze-dried nanocapsules Protamine NC (1% w/v) with a TCPH core, and loaded with rHBsAg at a 4:1 (protamine:rHBsAg) mass ratio were lyophilized (Labconco Corp, USA) in the presence of 5% (w/v) trehalose. For this purpose, the samples were frozen at -20 ºC, and then dried for two

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consecutive periods of 24 h in a high vacuum atmosphere, the first period at -35 ºC, and the second at 0 ºC. To end the process, the temperature was slowly increased to 25 ºC. The lyophilized product thus obtained was stored at 25 ºC (KBF 115 climate chamber, Binder; Germany) for 1, 3, 6, 9, and 12 months). At each time point, the formulations were resuspended in ultrapure water and their physicochemical characteristics tested. The integrity of the antigen was evaluated by Western blot (as described above).

Cytotoxicity of protamine nanocapsules in macrophages Cell toxicity was analyzed by the RTCA (real time cell analyser) xCELLigence equipment (ACEA Bioscience Inc., San Diego, CA), by the measurement of electrical impedance. Briefly, 1.5 X 104 cells/well were cultured in 16 well plates carrying gold electrodes, and incubated at 37 ºC in the presence of 5% CO2 until they reached exponential growth phase (18 h). Then, protamine NC with either TCPH or SQL at three different concentrations of protamine (25, 50, and 100 µg/ml) were added in duplicate, and the cells were incubated under the same conditions, for 48 h. Cells without NC, NC alone, and medium alone were used as controls. The whole experiment lasted 72 h, and the impedance, which correlates with the amount of attached cells, was monitored at 15 minutes intervals.

Internalization of protamine nanocapsules by macrophages In order to visualize the uptake of these protamine NC by phagocytic cells; a selected prototype of NC containing TCPH was prepared using fluorescently labeled protamine. For this purpose, 100 µl of 5-carboxytetramethylrodamine succinimidyl ester (TAMRA) solution in DMSO (10 mg/ml was slowly added to 1 ml of the protamine solution at 10 mg/ml in 0.1 M sodium

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bicarbonate buffer (pH 9.0) under stirring conditions. The resulting solution was incubated for 1 h at room temperature, and finally dialyzed (Slide- A Lyzer dialysis cassette 2000 MWCO, Thermo) for 48 h, to remove the free TAMRA. The protamine NC were prepared with this labeled protamine (Pr-TAMRA) following the procedure described previously. The stability of these NCs was confirmed by monitoring their size and PdI in RPMI medium for 48 h. The phagocytic assay was performed using the Raw 264.7 macrophage cell line. Briefly, 5 x 105 Raw 264.7 cells were plated in a 24-well plate (Falcon 3047, BD Biosciences, USA) with 1 ml of RPMI 10% FBS. TAMRA-labeled NC at a concentration of 50 µg/ml (concentration with respect to the amount of Pr-TAMRA associated to the NC), were added to the cells and incubated for 30 minutes. After three washes with PBS to remove non-internalized NC, the cells were observed under an inverted fluorescent microscope (IX50, Olympus Optical Co GmbH, Germany). The Raw 264.7 cells were also analyzed by flow cytometry. After incubation with the labeled NC, the cells were washed once with PBS, and then detached from the plate by incubation with 200 μL of Accutase (PAA, Austria) for 10 minutes at 37 ºC in the presence of 5% CO2. Finally, cells were transferred to a tube, washed with complete medium and centrifuged. The resulting suspension of cells in culture medium was analyzed using a flow cytometer (FC500, BeckmanCoulter, USA). The final confirmation of internalization of NC was performed using a confocal microscope (Leica SP5, Germany). Raw 264.7 cells were seeded on glass coverslips (Menzel-Gläser, Germany) in a 24-well plate, and incubated with NC as described above. After several washes, cells were fixed with 4% formaldehyde during 10 minutes, and then incubated with Alexa Fluor 488-phalloidin for 20 minutes in order to stain the cellular cytoskeleton. Finally, the coverslips were mounted over slides in the presence of ProLong Gold Antifade mounting medium

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(Invitrogen, USA) containing DAPI to stain the nucleus, and images were obtained with LAS AF software.

Cytokine secretion by peripheral blood mononuclear cells These studies were performed with peripheral blood mononuclear cells (PBMCs) of three independent healthy human donors. EDTA-anticoagulated blood was diluted with equal volume of PBS and centrifuged through a Ficoll-Hypaque (GE Healthcare Bio-Sciences, Uppsala, Sweden) gradient in a 7:3 (diluted blood : Ficoll) ratio at 260 x g for 30 minutes at 20 ºC, in order to separate mononuclear cells from the granulocyte and erythrocyte populations. Mononuclear cells were collected with a Pasteur pipette from the interface between Ficoll and plasma, and washed twice with complete medium by centrifugation (145 x g, 5 min, 20 ºC). For the assessment of cytokine production, 2 x 105 PBMCs were incubated during 24 h in 96well plates (37 ºC, 5% CO2) in the presence of protamine NC with either a SQL or TCPH core, at two concentrations (10 and 100 µg/ml, referred to the amount of protamine). As negative and positive control, cells were incubated with complete medium, or with 1 µg/ml of lipopolysaccharide (LPS) and 10 µg/ml phytohaemagglutinin (PHA), respectively. After 24 h, the plate was centrifuged (100 x g, 5 min, 4 ºC) and supernatants were collected and stored at -20 ºC until the analysis was performed. The secretion levels of different cytokines were determined using the human Th1/Th2 FlowCytomix assay (eBioscience, Austria), following the manufacturer’s instructions. Briefly, 25 μL of Ab-coated microspheres were incubated with 25 μL of the culture supernatants and 50 μL of biotin-conjugated secondary antibodies, for 2 h at room temperature (RT) on a microplate shaker. After several washes, 50 μL of streptavidin conjugated to phycoerythrin and 100 μL of

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PBS-T were added to the preparation and incubated for 1 h at RT on a microplate shaker. Finally, phycoerythrin-bound beads were studied by flow cytometry, and the data analyzed with the FlowCytomix Pro 3.0 Software (eBioscience, San Diego USA).

Study of complement activation by protamine nanocapsules The study of the complement cascade activation induced in vitro by protamine NC was performed by analyzing the C3 cleavage products by Western blot. For this purpose, human plasma was incubated with different concentrations of protamine NC (50-500 µg/ml range). The different dilutions were prepared by adding PBS (pH 7.4). Equal volumes of plasma, veronal buffer (pH 7.4), and protamine NC were added, mixed, and incubated at 37 ºC for 1 h. The positive (cobra venom factor) and negative (PBS) controls were prepared in the same conditions. The mixture was centrifuged (16000 x g, 0.5 h), and the supernatants run on a 10 % SDS-PAGE gel, and then transferred to a PVDF membrane (Immun-Blot, Biorad; Hercules, CA) using a transblot semidry transfer equipment (Bio-Rad; Hercules, CA). The PVDF membranes were blocked overnight with non-fat milk 5% (w/v) at 4 ºC, then incubated for 1 h in the presence of mouse mAb against human C3. After washing, secondary anti-mouse antibodies conjugated with alkaline phosphatase were added to the PVDF membranes for an additional hour of incubation. Finally, the membranes were revealed with 5-Bromo-4-chloro-3-indolyl-phosphate p-toluidine salt (BCIP).

Immunization studies upon intramuscular and intranasal administration Groups of 7 BALB/c female mice with an average weight of 20 g and 4 weeks of age were randomly assigned and immunized with the antigen-loaded formulations. The animals were kept conscious during the immunization and subsequent sample collection.

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Two groups of mice received two intramuscular (i.m.) administrations of rHBsAg loaded protamine NCs (0 and 4 week) with either a SQL or a TCPH oily core. Control mice received the conventional alum-adsorbed antigen, at a dose of 10 µg of rHBsAg, which was prepared in situ before immunization. For this purpose, the aluminum hydroxide and rHBsAg solutions were incubated in a 1:3 volume ratio for 30 min at 4 ºC under moderate agitation. The conjugate was then precipitated at 10000 x g for 10 min and resuspended in sterile PBS. On the other hand, two groups of mice (n = 7) received three doses (at 0, 4, and 16 weeks) of 10 µg rHBsAg associated to protamine NC (SQL or TCPH) by intranasal administration (i.n.), and another group received a combined (i.m.-i.n.) dose schedule of protamine NC with TCPH nucleus. This schedule consisted of a first dose (day 0) by the i.m. route, and two additional intranasal boosting doses at weeks 4 and 16. Serum samples were collected from the mouse maxillary vein at weeks 6, 18, and 26. A pool of sera for each group was prepared, and IgG endpoint titers for rHBsAg were determined by standard ELISA.

Quantification of rHBsAg specific IgG and its subtypes rHBsAg diluted in Carbonate Buffer (pH 9.6) at 5µg/ml was incubated overnight at 4ºC in Maxisorp 96-well plates. Then the plates were blocked with PBS-BSA 1% for 1 h at 37ºC in order to reduce non-specific interactions. To quantify anti rHBsAg IgG levels a mouse monoclonal IgG anti rHBsAg and a rabbit IgG anti rHBsAg of known concentrations were serially diluted and used to calculate IgG levels as µg/ml and mIU/ml. For this purpose, both control and serum samples were incubated for 2 h at 37ºC. Then, secondary Abs (goat anti-mouse IgG or anti-rabbit IgG

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conjugated to horseradish peroxidase) were added to each well and incubated for 1h at 37ºC. Finally, bound antibodies were revealed with ABTS and the IgG levels were calculated as mIU/ml. To analyze IgG subtypes (IgG1 and IgG2a), the pool of sera of the different groups of mice were analyzed following the same ELISA protocol as described above, but using goat anti-mouse IgG1 and IgG2a (conjugated to horseradish peroxidase) as secondary Abs for the calculation of the IgG1/IgG2a ratios.

Statistics The Kruskal-Wallis test was performed with the results of the in vivo studies using Statgraphic centurion XVI (Statpoint Technology). Differences were considered significant at a level of p < 0.05.

Ethics issues Institutional ethics approval to work with human samples from healthy donors was obtained from the Ethics Committee for Clinical Research (Xunta de Galicia, Spain. 2013/272). All participants included in the study gave their written informed consent. All protocols developed for animal experimentation complied with the guidelines of the Spanish regulations (Royal Decree 53/2013 and Law 6/2013) regarding the use of animals in scientific research and were approved by the Bioethical Committee of the University of Vigo.

RESULTS AND DISCUSSION Using influenza as a model antigenic protein, we recently reported the development of protamine nanocapsules (NC) that are efficient antigen delivery carriers.7 In this paper, we further explore

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the capacity of these nanocarriers for antigen delivery, when prepared with different oils that have demonstrated adjuvant capacity and recombinant hepatitis B antigen (rHBsAg), a complex lipoprotein antigen. These new nanocompositions were rigorously evaluated in vitro for their antigen loading, release properties, and stability profile. Within this context, one of our main goals was the development of a thermostable nanovaccine composition that would allow its long-term storage as a dry powder form. Furthermore, we have also studied the mechanistic aspects of the interaction of these NC with immunocompetent cells in vitro, and their ability to trigger specific immune responses against rHBsAg antigen in vivo. Development and characterization of protamine nanocapsules Protamine NC were developed, as described previously 7, through a comprehensive screening to select the optimal formulation variables, i.e. the most effective type of surfactants and oil:surfactant ratios. In this work, and with the purpose of further improve this technology, we considered two new compounds, squalene and α tocopherol, for the formation of the NC oily core. These two oils are present in several formulations already in the market, and their adjuvant capacity has been demonstrated in different clinical trials. Most importantly, both of them are components in adjuvants approved by the regulatory agencies, such as MF59 and ASO3.14 Table 1 shows that the NC compositions prepared with different oils have a nanometric size with low polydispersity index (PdI) and positive surface charge, being the latter due to the presence of a protamine shell around the oily core. The nature of the oil did not affect the particle size, but the surface charge was found to be different for both formulations. This difference could be attributed to the intrinsic characteristics of the oils used in the nucleus, which result in different zeta potential values of the nanoemulsions (i.e. oily core droplets without protamine) prepared with TCPH (-38±1 mV) or squalene (-17±1 mV).

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As shown in Figure 1, the NC are individual and homogeneous structures with spherical shape regardless of their core composition.

A

B

Figure 1: Transmission electron microscopy images of protamine nanocapsules. Formulations with squalene (A) or α tocopherol (B) as the oil in their nucleus. Scale bar: 1 µm.

Antigen association and release from protamine nanocapsules The recombinant hepatitis B surface antigen (rHBsAg) was associated to the NC by incubation at room temperature for up to 1 h. The rHBsAg is itself a virus-like protein nanoparticle with a size of 22 nm, and a negative surface charge.18, 19 These tiny nanoparticles could be easily attached to the NC’s surface by simple interaction with the positively charged guanidine groups of arginine, the main amino acid present in protamine. This interaction is supposed to be mediated by ionic forces, although other types of interactions such as hydrophobic and hydrogen bonds may also be involved in this process.20,

21

An evidence of this interaction is illustrated by the significant

decrease of the zeta potential of the antigen-loaded NC as compared to the corresponding blank formulations (Table 1).

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Table 1: Physicochemical characterization of protamine nanocapsules. Mean particle size, polydispersity index (PdI),  potential, and percentage of association of hepatitis B antigen to protamine NC with different core compositions. Data represents the mean ± SD, n=3. Size

Oil component

Squalene

α tocopherol

(nm)

PdI

 potential (mV)

% rHBsAg

Blank

215 ± 5

0.1

+18 ± 1

---

4:1 (NC:rHBsAg)

226 ± 19

0.2

+8 ± 3

82 ± 1

Blank

250 ± 23

0.2

+36 ± 3

---

4:1 (NC:rHBsAg)

210 ± 38

0.2

+20 ± 5

78 ± 13

The association of rHBsAg to the polymeric shell is a very attractive method to prepare antigenloaded protamine NC as it allows the exposition of the antigen on their surface, making it, thus, more accessible to antigen presenting cells (APC) and increasing the likeliness that it will eventually trigger an immune response. On the other hand, the results shown in Table 1 also indicate that the percentage of antigen associated to the NC, as determined by ELISA, is very high, irrespective of the nature of the oily core. These findings are in agreement with those found for chitosan NC where the association of the same antigen was near 70%, and a reduction of the positive zeta potential of the NC was also observed.22 In addition, protamine NC have previously shown high capacity to associate other types of antigens such as influenza hemagglutinin, a protein antigen.7 In order to evaluate the release of rHBsAg, we incubated protamine NC (TCPH nucleus) in PBS (pH 7.4) at 37 ºC for 24 h. Prior to these experiments, we confirmed that the physicochemical

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properties (size and PdI) of protamine NC were unaffected throughout the duration of the experiment (data not shown). Figure 2 shows the profile of the antigen release from the NC. Near 50% of rHBsAg is released within the first 24 h, which means that the antigen-protamine interaction in the formulation is reversible. This in vitro study also showed that the antigen was released slowly, without the pronounced burst effect typically observed for NC and nanoemulsions.23 This result suggests that protamine NC could behave as a reservoir of active molecules for controlled release in vivo. In addition, the results of the ELISA and Western blot analyses confirmed that the antigen maintains its integrity for the duration of the release process.

60

% HBsAg released

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40

20

0 0

5

10

15

20

25

Time (hours)

Figure 2: In vitro rHBsAg release from protamine nanocapsules. Formulation with TCPH as core in PBS (pH: 7.4) with Tween 80 (0.02 %) buffer after incubation at 37 ºC. Data represents the mean ± SD, n = 9.

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Stability of freeze-dried nanocapsules The fact that most vaccines are only stable at temperatures between 2 and 8 ºC poses a major concern in developing countries where the cold chain is often interrupted, resulting in a loss of the vaccine effectiveness.24 Improving vaccine stability at room temperature is therefore one of the greatest challenges in vaccine development. In order to assess the stability of rHBsAg-loaded protamine NC during storage, we first converted them to a freeze-dried powder. As seen in Figure 3.A, protamine NC with a TCPH core maintained their nanometric size and surface charge upon storage at room temperature (25 ºC) for up to one year. Interestingly, particle size was slightly decreased after the freeze-drying process. This phenomenon could be due to the presence of trehalose in the formulation as has been postulated that this kind of cryoprotectants can shield particles from one another during freeze drying. Besides, the lyophilization process itself could also cause the compaction of the nanocapsules, leading to this slight decrease when measuring particle size after reconstitution 25, 26.

Regarding the stability of the associated antigen, a Western blot image (Figure 3.B) of a sample at the 12 months time point shows that the antigen loaded onto protamine NC (lanes 2, 3, and 4) and stored at 25 °C did not lose its structure or antigenicity either during the freeze-drying process or over time. Lyophilized and non-lyophilized antigens (lane 5 and 6) stored at 25 °C and at 4 °C were used as respective controls. In this case it was possible to observe a relative loss of the amount of antigen confirming that the antigen preserves better its properties when it is associated to protamine NC. Overall, these results show that freeze-dried rHBsAg-loaded protamine NC are thermostable for at least 12 months. Other authors27 have reported NC with a hepatitis B antigen that were stable

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for one year; however, their formulation contained 7.5% propylene glycol, 40 mM phosphate, and 40 mM histidine in addition to the components of the original vaccine that included alum (aluminum hydroxide). Alum has important limitations when used as vaccine, such as being ineffective via non-parenteral routes, and also having unwanted side effects, i.e. local reactions and hypersensitization in allergic patients. The achievement of a thermostable vaccine formulation would be of great importance for public-sector stakeholders because of the economic impact this would have on public health.28 Moreover, a safe and thermostable vaccine would have a positive effect on the efficacy of immunization programs, reducing childhood mortality and morbidity in low-resource settings, such as developing countries.29

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Size (nm)

Size (nm)

A

Zeta Potential (mV)

450

45

350

30

250

15

150

 potential (mV)

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0 Before lyophilization

B

kDa

1

3

6

9

12

Time (months)

1

2

120 100 80 60 50

25 °C

3 25 °C

4

5

6

25 °C

25 °C

4 °C

40 30 rHBsAg 20

Figure 3: Stability of antigen loaded protamine nanocapsules as freeze dried powder. (A) Particle size and zeta potential of freeze-dried and reconstituted rHBsAg-loaded protamine NC (TCPH nucleus) stored at room temperature for different time periods (25 ºC) (n = 3 ± SD). (B) Western blot analysis of rHBsAg of samples after one year in storage. Lanes correspond to the molecular weight control (1), freeze dried protamine NC (2, 3, and 4) at 25°C, freeze-dried soluble antigen at 25 °C (5) and control of non-lyophilized antigen stored at 4°C (6). The amount of rHBsAg loaded was 1 µg in all cases.

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Interaction of protamine nanocapsules with macrophages: internalization and toxicity profile Recognition and internalization of nanocarriers by antigen presenting cells is an essential step for triggering immune responses.30 Therefore, in order to provide some insights on the mechanistic behavior of the NC described above, we studied cell viability after their internalization in a macrophage cell line (RAW 264.7). Figure 4 shows the exponential cell growth profile and the changes observed upon contact with protamine NC at different concentrations (25, 50, and 100 µg/ml, referred to the amount of protamine) for up to 48 h. Our results show that doses of 25 and 50 µg/ml did not interfere with the cell growth during the first 24 h. However, the exposure of the cells to a higher concentration (> 100 µg/ml) of NC affected the cell growth, and caused a decrease in their viability. These results are slightly different from those reported for chitosan NC using the same method. For example, chitosan NC doses of 25 and 50 µg/ml affected the viability of RAW 264.7 cells within the first 24 h of contact.20 Similar results were obtained by our group with high doses of blank NC (100 µg/ml): both prototypes (chitosan and protamine NC) decreased the cell viability at early times.22 Other studies using colorimetric techniques (e.g. MTT) have previously shown high cell viability with high concentrations of peptide and protein-loaded protamine NCs developed for oral administration.5,

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Altogether, these last studies agree with those reported

herein, showing that protamine NC do not have harmful effects on cell viability in a wide range of concentrations. In addition, possible differences when compared with other studies may depend on the experimental conditions selected. For example, in the current study, the results should be interpreted as a consequence of the long and continuous exposure of the cells to the NC. The results obtained for NC containing SQL were very similar to those presented above, thus indicating that the oily core did not significantly influence the cytotoxicity profile. These results

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were confirmed by the Quick Cell Proliferation Assay, a colorimetric method that measures the metabolic activity of living cells through the reduction of the added reagent (data not shown).

Figure 4: Toxicity profile of protamine nanocapsules. Effect of protamine NC (TCPH) on RAW 264.7 cells viability at 24 and 48 h, determined by the xCELLigence system (n = 3). Different concentrations of NC (25 µg/ml - blue, 50 µg/ml - green and 100 µg/ml - black) were studied. Red line corresponds to control cells (no NC). The vertical line indicates when NC were added to the cells (after 18 h of growth).

To study their cellular uptake, fluorescent NC (labeled with TAMRA) were incubated with RAW 264.7 cells for up to 30 min. Before incubation, we verified that the physicochemical characteristics of the NC (size, PdI, and zeta potential) were not affected by the use of TAMRAlabeled protamine (data not shown).

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A

B

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C

Figure 5: Internalization of TAMRA-labeled protamine nanocapsules. Formulations with TCPH core, at theoretical TAMRA-protamine concentration of 50 µg/ml in RAW 264.7 cells measured by (i) confocal microscopy: image of the maximum projection of a z-stack (A) and orthogonal section of the same z-stack (B) (green channel: Alexa fluor 488-phalloidin, blue channel: DAPI and red channel: Pr-TAMRA NC) and (ii) flow cytometry analysis (C). The confocal microscopy images, presented in Figure 5A-B, show that the fluorescent NC are located inside the cells and also attached to the cell membrane. In addition, using flow cytometry analysis (Figure 5C), we could observe that almost 100 % of the cells were positive for fluorescence after incubation with the protamine NC. In this sense, the positive surface charge of protamine is expected to play an important role on their cellular uptake32. The well-known membrane-translocating properties of protamine shared with polypeptides such as the Tat peptide9, could be responsible for this high internalization rate of the NC.

Cytokine secretion mediated by protamine nanocapsules in blood mononuclear cells The cytokine induction profile of protamine NC was evaluated in blood mononuclear cells at two different doses (10 and 100 µg/ml). The results summarized in Table 2 indicate that, regardless of the nature of the oily core, both types of NC could induce the production of a mixed pattern,

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involving mostly proinflammatory cytokines (IL-8, IL-1 β, and TNFα), but also IL-2 and TNFα (Th1 profile) and IL-4 and IL-6 (Th2 profile). Finally, some cytokines were not secreted at any concentration tested (IL-5 and IL-10). This mixed pattern of cytokine secretion is in agreement with those previously reported using protamine-condensed mRNA.33 Protamine, but not the mRNA alone, was able to activate blood mononuclear cells, and promote cytokine secretion. The same authors also found that protaminemRNA complexes can activate dendritic cells.34 In general, cationic particles are known to be better inductors of pro-inflammatory responses than anionic or neutral ones

35

and this could explain the capacity of protamine NC to induce

cytokine production by human PBMCs. In addition, it has been suggested that the particulated nature of vaccine nanocarriers, together with their high specific surface, may allow for high cellular uptake, antigen presentation and APC activation, resulting in increased adjuvant properties.36

Table 2: Cytokine production by human peripheral blood mononuclear cells upon incubation with protamine NCs containing TCPH (protamine NC TCPH), or squalene (protamine NC SQL) core, at two doses (10 and 100 µg/ml). The positive control was LPS (1 µg/ml) plus PHA. N/tested: number of responsive donors out of a total of 3. (10 µg/ml+: 1-10. ++: 10-100. +++: 100-1000. ++++: > 1000 fold higher than negative control (cells incubated in culture medium)).

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Concentration (µg/ml)

Th1 Profile

IL 12 IFN γ IL 2 TNF β

Th2 Profile

TNF α

Other proinflammatoy Cytokines

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

IL 10 IL4 IL5 IL 8 IL 1 β IL 6

10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100 10 100

Pr-NC TCPH Response N/tested + 1/3 ++ 1/3 ++ 1/3 + 1/3 + 1/3 + 1/3 + 1/3 ++ 1/3 ++ 1/3 + 2/3 + 2/3 + 1/3 + 3/3 ++ 2/3 ++ 2/3

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Pr-NC SQL LPS + PHA Response N/tested Response N/tested + 1/3 ++ 3/3 +++ 3/3 ++ 1/3 ++ 3/3 ++ 1/3 + 1/3 ++ 2/3 ++ 1/3 ++ 1/3 ++ 3/3 ++ 1/3 ++ 2/3 ++ 1/3 ++ 2/3 ++ 1/3 + 1/3 + 3/3 + 3/3 + 3/3 + 1/3 +++ 3/3 ++ 3/3 ++ 2/3 ++++ 3/3 ++ 2/3

Complement activation capacity of protamine nanocapsules The complement system is one of the most important components of the humoral innate immune system. It is composed by a number of proteins and cell surface receptors acting as a cascade, and it is involved in the recognition and clearance of invading agents, in mediating inflammation and promoting phagocytosis. It can be activated via three different pathways,37 all of which have in common the cleavage of the complement factor 3 (C3). Thus, the analysis of this cleavage is indicative of the complement activation by any of the three routes.

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Figure 6 shows that protamine NC containing either SQL or TCPH can induce C3 cleavage. This cleavage may be due to the interaction of the guanidine groups of the protamine present in the NC shell with the C3b α-chain.38 However, the intensity of this interaction was also affected by the nature of the oily core. Namely, TCPH-containing NC induced complement activation at a concentration of 50 µg/ml, whereas those containing SQL were only active at higher concentrations (> 100 µg/ml). Consequently, the complement activation capacity of protamine NC does not only depend on the protamine shell, but also on the type of immunostimulant oils present in the core. Pr NC TCPH

Pr NC SQL 50

100

250

500 Mw

-

CVF

kDa

50

100

250

500 Mw

-

CVF

250 130 95 72

C3 protein

55

C3 split products

36 26

11

Figure 6: Complement activation capacity of protamine nanocapsules. Western blot analysis of complement activation induced by protamine NC. Different concentrations of protamine NC (50, 100, 250, and 500 µg/ml according to protamine concentration) with SQL (left) or TCPH (right) were studied. Negative control was PBS (-) and positive control was cobra venom factor (CVF). Mw: molecular weight control. In the case of vaccine delivery carriers, complement activation could be indicative of their intrinsic capacity to stimulate the innate immune system, together with the promotion of specific

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responses through the activation of T and B cells.39 In fact, the activity of alum (the most widely used adjuvant in vaccines) is highly related to its ability to activate the complement cascade.40 Moreover, there is information in the literature showing the effect of particle size on the complement activation capacity. For example, Pedersen et al found that dextran-coated iron oxide core particles of a size around 250 nm induced greater complement activation than larger particles (600 nm).41 Overall, because of the synergistic effect of their size, structure, and composition on complement activation, protamine NC could be considered promising vaccine adjuvants due to their considerable capacity to interact with the immune system through different mechanisms, including complement activation.42

Immune response of rHBsAg-loaded protamine nanocapsules Encouraged by our findings that protamine NC have the capacity to combine an antigen with immunoadjuvant biomaterials (i.e. protamine and specific oils), our next goal was to assess their immune response upon in vivo administration, and to analyze the potential contribution of the oily core to the intensity of the response generated. More precisely, we analyzed the humoral immune response (anti rHBsAg serum IgG) elicited by these nanovaccines following either intramuscular (i.m.), intranasal (i.n.) or combined i.m./i.n. administration schedule in mice, using alum-adsorbed rHBsAg as positive control.

Immunization by the intramuscular route A 10 g dose of the formulations was administered at time zero and then again at week 4. The results shown in Figure 7A indicate that both NC formulations elicited steady antibody levels in the range of 100–1000 mIU/ml. These levels are far above those considered to be protective in

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humans (10 mIU/ml)43, and are of the same order of magnitude as those achieved by other nanovaccines containing the same antigen.22, 44 In contrast, the alum-adsorbed antigen induced an IgG response that was initially high but decreased over time, with antibody levels at the end of the study comparable to those obtained by our NC. Although the decrease of the antibody levels occurred in all formulations, it was more pronounced in the case of alum (Figure 7). With respect to the effect of the oily core, the statistical analysis of the data led us to conclude that protamine NC containing TCPH elicit a higher response than those containing SQL. This result is in agreement with the capacity of these systems to activate the complement cascade, since α tocopherol containing NC induced complement activation at lower doses than the squalene prototype. In addition, TCPH-containing protamine NC have higher zeta potential. This higher zeta potential probably promoted a better initial interaction with immunocompetent cells, contributing to a more efficient response.45 This confirms the results obtained by Morel et al, who studied the ASO3 adjuvant (based on α tocopherol and squalene) and concluded that the presence of α tocopherol increased cytokine secretion and that this component was essential for an optimal antibody response.46 However, it should be mentioned that the model antigen selected in this study, rHBsAg, might not be the one that benefits the most from our delivery technology, because this antigen interacts specifically with the phosphate groups of alum, increasing the adjuvant effect of this specific antigen. This effect would not occur with other vaccines.47 We could, thus, speculate that the benefit of this novel adjuvant carrier might be of greater value for other antigens for which alum does not give the adequate response.

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Immunization by the intranasal route Taking into account the positive surface charge of the protamine NC and, thus, their potential interaction with negatively charged mucosal surfaces, we also explored their capacity as antigen carriers for nasal immunization. The humoral IgG responses elicited upon nasal immunization with 3 doses of rHBsAg-loaded protamine NC are displayed in Figure 7B. The results obtained show significant and steady IgG levels throughout the study. Even though these levels were slightly lower than those achieved upon i.m. injection of the alum-adjuvanted antigen, they were above the threshold reported for a protective response in humans (10 mIU/ml). This protective response is in line with the results reported by our group for protamine-based nanoparticles and chitosan NC with rHBsAg as antigen.12,

20

The positive surface charge is a common parameter in these

formulations. In fact, the positive charge has been demonstrated to enhance the immune response, inducing the maturation and improving the function of antigen presenting cells.48 On the other hand, in line with the results obtained upon i.m. immunization, the formulation containing TCPH exhibited a higher response than the one containing SQL. Consequently, these results confirm the role of the oily core on the adjuvant activity of these NC. We also evaluated a combined i.m.–i.n. administration schedule consisting of one intramuscular dose followed by two intranasal doses. Interestingly, this protocol of administration led to a comparable response to the one obtained by the same formulation (TCPH nucleus) administered in two doses via the i.m. route. This result could be explained by the induction of a good adaptive immune response after the first intramuscular dose, generating memory cells which would have responded producing high antibody levels after the following intranasal boosting doses. Therefore, this schedule could be of high interest in cases where the response achieved by simple nasal administration does not reach the levels required for an adequate, long lasting protection.

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The possibility of a single dose of i.m. protamine NC administration followed by the needle-free administration of the boosting doses is very attractive as it could provide a significant reduction in the use of syringes, and improve patient compliance. This administration schedule, combined with the excellent thermostability of the formulation, could also help lower the costs of vaccination.

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Figure 7: Immune response of rHBsAg-loaded protamine nanocapsules. Serum specific IgG level of BALB/c mice immunized with protamine NC containing squalene (SQL) or α tocopherol (TCPH). (A) Serum IgG titers after 2 immunizations (weeks 0 and 4) with rHBsAg associated to NC or adsorbed on alum by intramuscular (i.m.) route. (B) Serum IgG titers after 3 immunizations (weeks 0, 4 and 16) with rHBsAg associated to NC by intranasal route (i.n.), or a combined

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schedule where the first dose was i.m. and the boosters were by i.n. against rHBsAg adsorbed to alum as a control (two i.m. doses at weeks 0 and 4). In all cases, the dose was 10 µg rHBsAg/mice. In both graphics all the values at each time point are significantly different from each other (p < 0.05).

Modulation of immune response In order to study the effect of the protamine NC on the modulation of the immune response, the levels of specific IgG1 and IgG2a anti-rHBAg antibodies were studied in the sera of mice immunized with the antigen-loaded nanocarriers (TCPH as oily core). The ratio of serum IgG1/IgG2a subtypes indicates the predominant type of immune response generated, either humoral or cellular response, mediated by T helper 2 (Th2) or T helper 1 (Th1) lymphocytes, respectively. The conclusion derived from our results is that the type of immune response elicited by protamine NC depends on the route of administration. This agrees with the results obtained for liposomes and trimethyl-chitosan nanoparticles encapsulating OVA, where IgG2a responses depended strongly on the route of vaccination after priming and boosting.49 Namely, following i.m. immunization the NC produced a strong and long-lasting Th2-type response, which is also the one typically observed for alum-based vaccines.50 This finding is in agreement with a previous study that evaluated various adjuvants in an influenza vaccine candidate. The results from this study showed that both AS03 (containing α-tocopherol) and alum induced a mainly humoral response. 51 On the other hand, following intranasal administration, we observed a predominant Th2 response at early time points, which evolved later towards a more balanced Th1/Th2 response (Figure 8).

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Finally, the combined i.m./i.n. administration schedule gave a comparable response to the one observed after i.m. administration alone. i.n.

12.00 10.00 Ratio IgG1/IgG2a

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i.m.

* * *

8.00

i.m./i.n.

HBsAg-Alum

* *

*

6.00

*

4.00 2.00 0.00 6

18 Time (weeks)

26

Figure 8: Modulation of the immune response by protamine nanocapsules. Comparison of the IgG subtype ratio (IgG1/IgG2a) elicited by protamine NC upon intramuscular (i.m.), nasal (i.n.) or combined (i.m./i.n.) administration schedule. The ratio of alum-adsorbed rHBsAg (ALUM i.m.) is shown as control. (p < 0.05).

The predominantly humoral response elicited by the antigen-loaded nanocarriers might be attributed to different patterns of interaction of the NC with the antigen presenting cells and naïve Th cells.52 These results confirm our findings regarding the cytokine profile after the in vitro activation of peripheral blood mononuclear cells (Table 2) and are also in accordance with those reported for liposomes and trimethyl-chitosan nanoparticles.49

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Altogether, these results suggest the potential of protamine NC as adjuvants for parenteral and nasal administration. Moreover, the possibility of administering a single dose by parenteral route followed by a self-administered nasal boosting is a positive development because it makes possible to combine both modalities of administration, and thus achieve both, humoral and cellular, responses.

CONCLUSIONS We report here the development of new thermostable antigen delivery vehicles, protamine NC, which may be an interesting alternative to currently available vaccine adjuvants because they (i) allow for the simultaneous loading of antigens and immunomodulators such as oils and oily soluble compounds; (ii) preserve the stability of the associated antigen for at least one year at room temperature; (iii) have the ability to stimulate human immune cells and promote the secretion of cytokines that are relevant for immunostimulation and (iv) elicit humoral and cellular responses against the associated antigen following i.m. and nasal administration. In conclusion, protamine NC allow for the formulation of challenging antigens for which a mixed Th1/Th2 response might be required. Furthermore, they represent a promising option for needle-free vaccination strategies, and for the preservation of the antigens stability at room temperature. Current and future studies in our laboratory, will focus on whether this novel technology is also a possibility for other antigens.

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ACKNOWLEDGEMENTS This work was supported by the Spanish Ministry of Economy and Competitivenes (SAF201130337-C02-02 and BIO2014-53091-C3-1-R), the FP7/REGPOT-2012- 2013.1-BIOCAPS316265, the Consellería de Cultura, Educación e Ordenación Universitaria (Competitive Reference Groups and Centro singular de investigación de Galicia, ED431G/05) and the European Regional Development Fund (ERDF).We would like to thank Jesús Méndez from CACTI facilities of the University of Vigo and the technical assistance of Rafael Romero. JV González-Aramundiz and M. Peleteiro Olmedo acknowledge their fellowships from the Presidente de la Republica program (Chile) and Spanish Ministry of Education (FPU predoctoral grant) respectively.

REFERENCES 1. González-Aramundiz, J. V.; Cordeiro, A. S.; Csaba, N.; De la Fuente, M.; Alonso, M. Nanovaccine: nanocarriers for antigen delivery. Biologie Aujourd'hui 2012, 206, (4), 249-261. 2. Cordeiro, A. S.; Alonso, M. J.; de la Fuente, M. Nanoengineering of vaccines using natural polysaccharides. Biotechnol. Adv. 2015, 33, (6, Part 3), 1279-1293. 3. González-Aramundiz, J. V.; Lozano, M. V.; Sousa-Herves, A.; Fernandez-Megia, E.; Csaba, N. Polypeptides and polyaminoacids in drug delivery. Expert Opin. Drug Deliv. 2012, 9, (2), 183-201. 4. Reed, S. G.; Orr, M. T.; Fox, C. B. Key roles of adjuvants in modern vaccines. Nat. Med. 2013, 19, (12), 1597-1608. 5. Thwala, L. N.; Beloqui, A.; Csaba, N. S.; González-Touceda, D.; Tovar, S.; Dieguez, C.; Alonso, M. J.; Préat, V. The interaction of protamine nanocapsules with the intestinal epithelium: A mechanistic approach. J. Control. Release 2016, 243, 109-120. 6. Reimondez-Troitiño, S.; Alcalde, I.; Csaba, N.; Íñigo-Portugués, A.; de la Fuente, M.; Bech, F.; Riestra, A. C.; Merayo-Lloves, J.; Alonso, M. J. Polymeric nanocapsules: a potential new therapy for corneal wound healing. Drug Deliv Transl Res. 2016, 6, (6), 708-721. 7. González-Aramundiz, J. V.; Presas, E.; Dalmau-Mena, I.; Martínez-Pulgarín, S.; Alonso, C.; Escribano, J. M.; Alonso, M. J.; Csaba, N. S. Rational design of protamine nanocapsules as antigen delivery carriers. J. Control. Release 2017, 245, 62-69. 8. He, H. N.; Ye, J. X.; Liu, E. G.; Liang, Q. L.; Liu, Q.; Yang, V. C. Low molecular weight protamine (LMWP): A nontoxic protamine substitute and an effective cell-penetrating peptide. J. Control. Release 2014, 193, 63-73.

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Molecular Pharmaceutics

9. Reynolds, F.; Weissleder, R.; Josephson, L. Protamine as an Efficient MembraneTranslocating Peptide. Bioconjug. Chem. 2005, 16, (5), 1240-1245. 10. Martínez Gómez, J. M.; Csaba, N.; Fischer, S.; Sichelstiel, A.; Kündig, T. M.; Gander, B.; Johansen, P. Surface coating of PLGA microparticles with protamine enhances their immunological performance through facilitated phagocytosis. J. Control. Release 2008, 130, (2), 161-167. 11. Zeng, Z.; Dai, S.; Jiao, Y.; Jiang, L.; Zhao, Y.; Wang, B.; Zong, L. Mannosylated protamine as a novel DNA vaccine carrier for effective induction of anti-tumor immune responses. Int. J. Pharm. 2016, 506, (1–2), 394-406. 12. González-Aramundiz, J. V.; Peleteiro Olmedo, M.; González-Fernández, Á.; Alonso Fernández, M. J.; Csaba, N. S. Protamine-based nanoparticles as new antigen delivery systems. Eur. J. Pharm. Biopharm. 2015, 97, Part A, 51-59. 13. Pekmezci, D., Vitamin E and immunity. In Vitamins and the Immune System, Litwack, G., Ed. Elsevier Academic Press Inc: San Diego, 2011; Vol. 86, pp 179-215. 14. Correia-Pinto, J. F.; Csaba, N.; Alonso, M. J. Vaccine delivery carriers: Insights and future perspectives. Int. J. Pharm. 2013, 440, (1), 27-38. 15. Schillie, S.; Vellozzi, C.; Reingold, A.; Harris, A.; Haber, P.; Ward, J. W.; Nelson, N. P. Prevention of Hepatitis B Virus Infection in the United States: Recommendations of the Advisory Committee on Immunization Practices. Recommendations of the Advisory Committee on Immunization Practices 2018, 67, ((No. RR-1)), 1-31. 16. Kristensen, D.; Chen, D. Strategies to advance vaccine technologies for resource-poor settings. Vaccine 2013, 31, Supplement 2, (0), B157-B162. 17. Vicente, S.; Diaz-Freitas, B.; Peleteiro, M.; Sanchez, A.; Pascual, D. W.; GonzalezFernandez, A.; Alonso, M. J. A Polymer/Oil Based Nanovaccine as a Single-Dose Immunization Approach. PLoS ONE 2013, 8, (4), e62500. 18. Okeefe, D. O.; Paiva, A. M. Assay for recombinant hepatitis B surface antigen using reversed-phase high-performance liquid chromatography. Anal. Biochem. 1995, 230, (1), 48-54. 19. Gavilanes, F.; Gonzalez-Ros, J. M.; Peterson, D. L. Structure of hepatitis B surface antigen. Characterization of the lipid components and their association with the viral proteins. J. Biol. Chem. 1982, 257, (13), 7770-7. 20. Vicente, S.; Peleteiro, M.; Díaz-Freitas, B.; Sanchez, A.; González-Fernández, Á.; Alonso, M. J. Co-delivery of viral proteins and a TLR7 agonist from polysaccharide nanocapsules: A needle-free vaccination strategy. J. Control. Release 2013, 172, (3), 773-781. 21. Bussio, J.; Molina-Perea, C.; González-Aramundiz, J. Lower-Sized Chitosan Nanocapsules for Transcutaneous Antigen Delivery. Nanomaterials 2018, 8, (9), 659. 22. Vicente, S.; Peleteiro, M.; Gonzalez-Aramundiz, J. V.; Diaz-Freitas, B.; MartinezPulgarin, S.; Neissa, J. I.; Escribano, J. M.; Sanchez, A.; Gonzalez-Fernandez, A.; Alonso, M. J. Highly versatile immunostimulating nanocapsules for specific immune potentiation. Nanomed. 2014, 9, (15), 2273-2289. 23. Gehrcke, M.; Giuliani, L. M.; Ferreira, L. M.; Barbieri, A. V.; Sari, M. H. M.; da Silveira, E. F.; Azambuja, J. H.; Nogueira, C. W.; Braganhol, E.; Cruz, L. Enhanced photostability, radical scavenging and antitumor activity of indole-3-carbinol-loaded rose hip oil nanocapsules. Mater Sci Eng C Mater Biol Appl. 2017, 74, (Supplement C), 279-286. 24. Kristensen, D. D.; Lorenson, T.; Bartholomew, K.; Villadiego, S. Can thermostable vaccines help address cold-chain challenges? Results from stakeholder interviews in six low- and middle-income countries. Vaccine 2016, 34, (7), 899-904.

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Page 38 of 39

25. Allison, S. D.; Molina, M. d. C.; Anchordoquy, T. J. Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis. Biochimica et Biophysica Acta (BBA) - Biomembranes 2000, 1468, (1), 127-138. 26. Park, K. Prevention of nanoparticle aggregation during freeze-drying. J. Control. Release 2017, 248, 153. 27. Braun, L. J.; Jezek, J.; Peterson, S.; Tyagi, A.; Perkins, S.; Sylvester, D.; Guy, M.; Lal, M.; Priddy, S.; Plzak, H.; Kristensen, D.; Chen, D. Characterization of a thermostable hepatitis B vaccine formulation. Vaccine 2009, 27, (34), 4609-4614. 28. Zehrung, D.; Jarrahian, C.; Giersing, B.; Kristensen, D. Exploring new packaging and delivery options for the immunization supply chain. Vaccine 2017, 35, (17), 2265-2271. 29. Levin, A.; Levin, C.; Kristensen, D.; Matthias, D. An economic evaluation of thermostable vaccines in Cambodia, Ghana and Bangladesh. Vaccine 2007, 25, (39), 6945-6957. 30. Benne, N.; van Duijn, J.; Kuiper, J.; Jiskoot, W.; Slütter, B. Orchestrating immune responses: How size, shape and rigidity affect the immunogenicity of particulate vaccines. J. Control. Release 2016, 234, 124-134. 31. Jakubiak, P.; Thwala, L. N.; Cadete, A.; Préat, V.; Alonso, M. J.; Beloqui, A.; Csaba, N. Solvent-free protamine nanocapsules as carriers for mucosal delivery of therapeutics. Eur. Polym. J. 2017, 93, 695-705. 32. Peleteiro, M.; Presas, E.; González-Aramundiz, J. V.; Sánchez-Correa, B.; SimónVázquez, R.; Csaba, N.; Alonso, M. J.; González-Fernández, Á. Polymeric Nanocapsules for Vaccine Delivery: Influence of the Polymeric Shell on the Interaction With the Immune System. Front. Immunol. 2018, 9, (791). 33. Scheel, B.; Braedel, S.; Probst, J.; Carralot, J. P.; Wagner, H.; Schild, H.; Jung, G.; Rammensee, H. G.; Pascolo, S. Immunostimulating capacities of stabilized RNA molecules. Eur. J. Immunol. 2004, 34, (2), 537-547. 34. Scheel, B.; Teufel, R.; Probst, J.; Carralot, J.-P.; Geginat, J.; Radsak, M.; Jarrossay, D.; Wagner, H.; Jung, G.; Rammensee, H.-G.; Hoerr, I.; Pascolo, S. Toll-like receptor-dependent activation of several human blood cell types by protamine-condensed mRNA. Eur. J. Immunol. 2005, 35, (5), 1557-1566. 35. Dobrovolskaia, M. A.; McNeil, S. E. Immunological properties of engineered nanomaterials. Nat. Nanotechnol. 2007, 2, (8), 469-478. 36. Neek, M.; Tucker, J. A.; Kim, T. I.; Molino, N. M.; Nelson, E. L.; Wang, S.-W. Codelivery of human cancer-testis antigens with adjuvant in protein nanoparticles induces higher cell-mediated immune responses. Biomaterials 2018, 156, (Supplement C), 194-203. 37. Alcorlo, M.; Lopez-Perrote, A.; Delgado, S.; Yebenes, H.; Subias, M.; RodriguezGallego, C.; de Cordoba, S. R.; Llorca, O. Structural insights on complement activation. FEBS J. 2015, 282, (20), 3883-3891. 38. Moghimi, S. M.; Andersen, A. J.; Ahmadvand, D.; Wibroe, P. P.; Andresen, T. L.; Hunter, A. C. Material properties in complement activation. Adv. Drug Delivery. Rev. 2011, 63, (12), 1000-1007. 39. Reddy, S. T.; van der Vlies, A. J.; Simeoni, E.; Angeli, V.; Randolph, G. J.; O'Neill, C. P.; Lee, L. K.; Swartz, M. A.; Hubbell, J. A. Exploiting lymphatic transport and complement activation in nanoparticle vaccines. Nat. Biotechnol. 2007, 25, (10), 1159-1164. 40. Gupta, R. K. Aluminum compounds as vaccine adjuvants. Adv. Drug Delivery. Rev. 1998, 32, (3), 155-172.

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41. Pedersen, M. B.; Zhou, X.; Larsen, E. K. U.; Sørensen, U. S.; Kjems, J.; Nygaard, J. V.; Nyengaard, J. R.; Meyer, R. L.; Boesen, T.; Vorup-Jensen, T. Curvature of Synthetic and Natural Surfaces Is an Important Target Feature in Classical Pathway Complement Activation. The Journal of Immunology 2010, 184, (4), 1931-1945. 42. Liu, Y.; Yin, Y.; Wang, L. Y.; Zhang, W. F.; Chen, X. M.; Yang, X. X.; Xu, J. J.; Ma, G. H. Engineering Biomaterial-Associated Complement Activation to Improve Vaccine Efficacy. Biomacromolecules 2013, 14, (9), 3321-3328. 43. CDC. Hepatitis B. Center for Disease Control and Prevention 2012. 44. Tafaghodi, M.; Saluja, V.; Kersten, G. F. A.; Kraan, H.; Slütter, B.; Amorij, J.-P.; Jiskoot, W. Hepatitis B surface antigen nanoparticles coated with chitosan and trimethyl chitosan: Impact of formulation on physicochemical and immunological characteristics. Vaccine 2012, 30, (36), 5341-5348. 45. Christensen, D.; Korsholm, K. S.; Andersen, P.; Agger, E. M. Cationic liposomes as vaccine adjuvants. Expert Rev. Vaccines 2011, 10, (4), 513-521. 46. Morel, S.; Didierlaurent, A.; Bourguignon, P.; Delhaye, S.; Baras, B.; Jacob, V.; Planty, C.; Elouahabi, A.; Harvengt, P.; Carlsen, H.; Kielland, A.; Chomez, P.; Garcon, N.; Van Mechelen, M. Adjuvant System AS03 containing alpha-tocopherol modulates innate immune response and leads to improved adaptive immunity. Vaccine 2011, 29, (13), 2461-2473. 47. Ghimire, T. R. The mechanisms of action of vaccines containing aluminum adjuvants: an in vitro vs in vivo paradigm. SpringerPlus 2015, 4, 181. 48. Ma, Y.; Zhuang, Y.; Xie, X.; Wang, C.; Wang, F.; Zhou, D.; Zeng, J.; Cai, L. The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccineinduced immune responses. Nanoscale 2011, 3, (5), 2307-2314. 49. Mohanan, D.; Slütter, B.; Henriksen-Lacey, M.; Jiskoot, W.; Bouwstra, J. A.; Perrie, Y.; Kündig, T. M.; Gander, B.; Johansen, P. Administration routes affect the quality of immune responses: A cross-sectional evaluation of particulate antigen-delivery systems. J. Control. Release 2010, 147, (3), 342-349. 50. Brewer, J. M. (How) do aluminium adjuvants work? Immunol. Lett. 2006, 102, (1), 1015. 51. Baz, M.; Samant, M.; Zekki, H.; Tribout-Jover, P.; Plante, M.; Lanteigne, A.-M.; Hamelin, M.-E.; Mallett, C.; Papadopoulou, B.; Boivin, G. Effects of Different Adjuvants in the Context of Intramuscular and Intranasal Routes on Humoral and Cellular Immune Responses Induced by Detergent-Split A/H3N2 Influenza Vaccines in Mice. Clin. Vaccine Immunol. 2012, 19, (2), 209-218. 52. Kaiko, G. E.; Horvat, J. C.; Beagley, K. W.; Hansbro, P. M. Immunological decisionmaking: how does the immune system decide to mount a helper T-cell response? Immunology 2008, 123, (3), 326-338.

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