Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties

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International Journal of Pharmaceutics xxx (2015) xxx–xxx

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Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties C. Gamazoa,* , H. Bussmannb , S. Giemsab , A.I. Camachoa , Daisy Unsihuayc , N. Martín-Arbellad , J.M. Irached a

Department of Microbiology, University of Navarra, 31008 Pamplona, Spain Department of Pharmacy, University of Bonn, 53115 Bonn, Germany Departament of Chemistry, Universidad Nacional de Ingeniería, Lima 25, Lima, Perú d Department of Pharmacy and Pharmaceutical Technology, University of Navarra, 31008 Pamplona, Spain b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 5 August 2015 Received in revised form 5 October 2015 Accepted 7 October 2015 Available online xxx

Understanding how nanoparticles are formed and how those processes ultimately determine the nanoparticles’ properties and their impact on their capture by immune cells is key in vaccination studies. Accordingly, we wanted to evaluate how the previously described poly (anhydride)-based nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride (NP) interact with macrophages, and how this process depends on the physicochemical properties derived from the method of preparation. First, we studied the influence of the desolvation and drying processes used to obtain the nanoparticles. NP prepared by the desolvation of the polymers in acetone with a mixture of ethanol and water yielded higher mean diameters than those obtained in the presence of water (250 nm vs. 180 nm). In addition, nanoparticles dried by lyophilization presented higher negative zeta potentials than those dried by spray-drying ( 47 mV vs. 35 mV). Second, the influence of the NP formulation on the phagocytosis by J774 murine macrophage-like cell line was investigated. The data indicated that NPs prepared in the presence of water were at least three-times more efficiently internalized by cells than NPs prepared with the mixture of ethanol and water. Besides, lyophilized nanoparticles appeared to be more efficiently taken up by J744 cells than those dried by spray-drying. To further understand the specific mechanisms involved in the cellular internalization of NPs, different pharmacological inhibitors were used to interfere with specific uptake pathways. Results suggest that the NP formulations, particularly, nanoparticles prepared by the addition of ethanol:water, are internalized by the clathrin-mediated endocytosis, rather than caveolae-mediated mechanisms, supporting their previously described vaccine adjuvant properties. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Nanoparticle Macrophage Antigen presenting cell Cellular uptake Clathrin

1. Introduction Vaccine strategies can be grouped into broadly two fundamental approaches: live-attenuated and non-living vaccines (e.g., bacterins and subunits vaccines). Recent strategies involve the use of purified or recombinant subunit vaccines since they offer the safest alternative in vaccination. However, they suffer from a low immunogenicity and, in general additional components are required to confer protective immunity. This led us to the full concept of vaccine formulation, enclosing antigens but also other chemical substances with adjuvant properties (Brito et al., 2013). The development of adjuvants has been mainly based on an empirical approach and, as a consequence, their mechanisms of

* Corresponding author. Fax: +34 9 48 42 56 49. E-mail address: [email protected] (C. Gamazo).

action are still not completely known, precluding their extensive applicability. Furthermore, this erratic comprehension of their effects leads to several safety concerns that limit their use. The understanding of the immune mechanisms will support the rational design of adjuvants filling the still remaining gaps (Levitz and Golenbock, 2012). Among several strategies, the employment of nanoparticles appears as a potential tool since they put together immunological properties as well as technical and practical features. Nanoparticles offer several advantages, including their capability as a delivery system, offering protection and controlled release properties of the loaded antigen (Gamazo et al., 2015). In addition, the particulate nature of nanoparticles has some inherent ability to facilitate antigen cross-presentation by antigen presenting cells (APCs). In this context, poly(anhydride)-based nanoparticles of the copolymer of methyl vinyl ether and maleic anhydride have been shown to modulate the immune response (Gomez et al., 2006, 2007; Irache et al., 2010; Kipper et al., 2006;

http://dx.doi.org/10.1016/j.ijpharm.2015.10.030 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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Mallapragada and Narasimhan, 2008; Ochoa et al., 2007; Salman et al., 2005, 2009). In fact, previous results in our group have shown that antigenic complexes loaded in poly(anhydride) nanoparticles shift the immune response from Th2 to Th1 (De S. Rebouças et al., 2012, 2014; Gomez et al., 2007; Irache et al., 2010; Ochoa et al., 2007). The mechanism of this immunomodulation has been partially elucidated, although there still remain gaps to full understanding. It has been demonstrated that these nanoparticles act as agonists of TLR2 and TLR4 (Tamayo et al., 2010) and they are capable of activating the complement system by targeting C3b (Camacho et al., 2011; Tamayo et al., 2010). Now, we wanted to go further in the understanding of how particle properties affect cellular uptake and/or interaction. To this respect, the rate of uptake and intracellular localization of a variety of nanoparticles has been studied by many research groups, and several review articles summarizing the published data are available (Hild et al., 2008; Hillaireau and Couvreur, 2009; Iversen et al., 2011; Mailander and Landfester, 2009; Maysinger et al., 2007; Delehanty et al., 2009; Sahay et al., 2010; Verma and Stellacci, 2010). These reviews expose the difficulty to draw general conclusions about how to produce particles for optimal cellular uptake, as the rate and mechanism of uptake turns out to be cell-type and density dependent (Kaplan, 1976; Snijder et al., 2009) and vary between nanoparticles with different size, surface charge, material compositions and other surface properties. Still, the study of how nanoparticles are captured by certain cells is of particular relevance considering that the different mechanisms of internalization may trigger different immune responses (Mogensen, 2009). Accordingly, we wanted to evaluate how the previously described poly(anhydride) nanoparticles interact with immune cells, such as macrophages, depending on the physicochemical properties derived from the method of preparation of these nanoparticles. Thus, the aspects studied here are the employment of different methods for nanoparticle preparation and the subsequent effect on size and zeta-potential. Subsequently, we explored the interaction between four different formulations based on the copolymer of methyl vinyl ether and maleic anhydride with J744 macrophage cell line by measuring cytotoxicity, the overall uptake kinetic, and the mechanisms of uptake by using selected uptake inhibitors.

2.2. Preparation of poly(anhydride) nanoparticles Poly(anhydride) nanoparticles were obtained by a modification of the solvent displacement method previously described (Arbós et al., 2002), followed by a purification step by ultracentrifugation and, finally dried by either lyophilization or spray-drying. Briefly, a 2% w/v solution of the copolymer of methyl vinyl ether and maleic anhydride (PVM/MA) in acetone was prepared under magnetic stirring at room temperature. Nanoparticles were formed by the addition of 2 volumes of acetone to either water or to a mixture of ethanol and water (1:1 by volume). Freshly formed nanoparticles were collected by centrifugation (27.000  g, 20 min, 4  C). Supernatants were discarded and, depending on the drying procedure, the pellets redispersed in an aqueous solution containing either sucrose (5% w/v) or mannitol (5% w/v). Then, nanoparticles dispersed in the sucrose solution were dried using a freeze–dryer apparatus (VirTis, New York, U.S.A.). On the other hand, nanoparticles dispersed in the mannitol solution were dried in a Büchi Mini Spray Drier B-290 apparatus (BüchiLabortechnik AG, Switzerland) under the following experimental conditions: (i) inlet temperature of 90  C, (ii) outlet temperature 45–50  C, (iii) air pressure: 2–5 bar, (iv) pumping rate of 2–6 mL/min, (v) aspirator of 100% and (vi) air flow at 900 L/h. Nanoparticles were also fluorescently labeled with Lumogen red. For this purpose, Lumogen was dissolved in the acetone phase (0.02% w/v) containing the polymer. Then, nanoparticles were obtained, purified and dried as described above. The different sets of nanoparticles are identified in Table 1. 2.3. Characterization of nanoparticles The particle size and the zeta potential of nanoparticles were determined by photon correlation spectroscopy (PCS) and electrophoretic laser Doppler anemometry, respectively, using a Zetamaster analyzer system (Malvern Instruments Ltd., Worcestershire, UK). The diameter of the nanoparticles was determined after dispersion in ultrapure water and measured at 25  C by dynamic light scattering angle of 90 . The zeta potential was determined as follows: 200 mL of the samples was diluted in 2 mL of a 0.1 mM KCl solution adjusted to pH 7.4.

2. Material and methods 2.4. Cell culture 2.1. Chemicals Poly(methyl vinyl ether-co-maleic anhydride) or poly(anhydride) (Gantrez1 AN 119; MW 200,000) was kindly gifted by ISP (Barcelona, Spain). Lumogen red was supplied by Kremer Pigmente (Germany). MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5 diphenyltetrazolium bromide), phorbol-12-myristate-13-acetate (PMA), chlorpromazine hydrochloride, sodium azide, genestein and cytochalasin D were provided by Sigma-Life Science (Germany). Wortmannin and LY94002 were obtained from Cell Signaling Technology (USA). Dihydrorhodamine (DHR) from Life Technologies (USA). Dimethylsulfoxid from Panreac (Spain). Saccharose and mannitol were supplied by Guinama (Spain).

Mouse macrophage J744 cells (passage 4–10 from freezing stocks in liquid nitrogen) were grown at 37  C (5% CO2 and 95% humidified air) in cell medium [RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/ streptomycin (invitrogen)]. 2.5. MTT-cytotoxicity assay J774 cells were used for in vitro cytotoxicity analysis of the prepared PVM/MA nanoparticles using the MTT-colorimetric monocyte mediated cytotoxicity assay, based upon the ability of living cells to reduce 3-[4,5-dimethylthiazol-2-yl]-2,

Table 1 Nanoparticles used in this study.

Spray-dried nanoparticles prepared in an ethanol:water mixture Spray-dried nanoparticles prepared in water Lyophilized nanoparticles prepared in an ethanol:water mixture Lyophilized GantrezTM nanoparticles prepared in water

Empty nanoparticles

Lumogen-loaded nanoparticles

SD EtOH SD H2O LF EtOH LF H2O

SD EtOH L SD H2O L LF EtOH L LF H2O L

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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5 diphenyltetrazolium bromide (MTT) into formazan (Promega, USA). A total of 5  104 J774 cells were seeded in a 96 well plate (24 h, 37  C, 5% CO2, 95% humidified air). After washing with PBS, nanoparticles suspended in cell medium were added to the wells to reach a final concentration of 25, 100, 200 or 400 mg/mL. As a positive control, cytotoxic Triton X100 in cell medium (0.5 % v/v) was used. Cells were incubated for 3 h or 24 h. After incubation, medium was removed and 100 mL of MTT solution was added (3 h, 37  C, 5% CO2, 95% humidified air). Then, 200 mL of dimethyl sulfoxide was added to dissolve the reduced formazan product generated by live cells. To determine the relative viability of the cells incubated with nanoparticles to untreated cells, the formazan absorbance was measured at 540 nm. 2.6. Respiratory burst Cells were seeded in 96/well flat bottomed microtiter plate with a number of 2  104 cells per well in 100 mL of cell medium. Cells were checked under a microscope for confluence. After 24 h of incubation at 37  C (5% CO2 and 95% humidified air) the cells demonstrated a confluence of 70%. Thereupon, the supernatants were removed and the cells were washed with 50 mL of PBS. Then, cells were pre-incubated for 15 min with dihydrorhodamine-123 (DHR) 5 mg/mL. Oxidation of DHR to fluorescent rhodamine is a marker of cellular oxidant production. After this pre-incubation, cells were washed with 100 mL of PBS. Next, nanoparticles (final concentration 100 mg/ml) were added and incubated for another 3 h. Negative controls, including cells without the presence of DHR and/or nanoparticles, were also carried out. As a positive control PMA (10 mg/mL) diluted in cell medium was used. After this incubation time, cells were then washed 3 times with 100 mL of PBS and afterwards treated with 50 mL of trypsin for 2 min at 37  C (5% CO2, 95% humidified air). To support detachment of the adherent cells the plate was clapped gently. Then, it was added 50 mL of cell medium and the suspension was carefully pumped up and down to disperse the cells homogeneously. The plate was centrifuged (300  g, 5 min), the supernatant removed, and cells were resuspended in 150 mL of PBS containing 1% (v/v) BSA. Thus, cells were suspended in a final volume of 300 mL. The samples were measured by flow cytometry using an Attune acoustic focusing cytometer (Applied Biosystems). Macrophages were recognized on the basis of forward-angle light scatter (FS) and side-angle light scatter (SS). 2.7. Cellular uptake of nanoparticles Cells were seeded in a 96 well-flat bottomed microtiter plate (2  104 cells per well in 100 mL of cell medium). After 24 h of incubation (37  C in 5% CO2 and 95 % humidified air) the cells demonstrated a confluence of 70%. Thereupon the supernatants were removed and the cells were washed with 50 mL of PBS. Then 100 mL of lumogen containing nanoparticles in cell medium at 25, 100, 200 or 400 mg/mL were added, and cells were incubated for another 3 h or 24 h.

3

After this incubation time, the cells were washed 3 times with 100 mL of PBS and treated with 50 mL of trypsin for 2 min (37  C, 5% CO2, 95% humidified air). To support detachment of the adherent cells the plate was clapped gently. It was added 50 mL of cell medium and the suspension was carefully pumped up and down to disperse the cells homogeneously. The plate was centrifuged (300  g, 5 min) and the supernatant was removed carefully. It was added 150 mL of PBS containing 1% BSA and cells were resuspended to be analysed by flow cytometry. 2.8. Inhibition of endocytic pathways Cells were seeded in a 96/well flat bottomed microtiter plate with a number of 2  104 cells per well in 100 mL of cell medium. After 24 h of incubation (37  C in 5% CO2 and 95% humidified air) the cells demonstrated a confluence of 70%. Thereupon the supernatants were removed and the cells were washed with 50 mL of PBS. Cells were pre-incubated for 30 min with the different drugs at the following concentrations: Cytochalasin D, 5 mM; Chlorpromazine, 1 mg/mL; LY294002, 50 mM; Wortmannin, 1 mM. The dilutions were performed in cell medium. After this pre-incubation, nanoparticles in cell medium (final concentration, 100 mg/mL) were added and cells were incubated for another 3 or 24 h. The cells were then washed three times with 100 mL of PBS, treated with 50 mL of trypsin, washed and resuspended as above before being analysed by flow cytometry. 2.9. Statistical analysis GraphPad Prism 5 statistical analysis software was used in all statistical analyses performed in this study (GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California, USA). All results have been presented as mean  standard deviation (SD). Statistical comparisons were conducted using one-way analysis of variance (ANOVA), followed by the Dunnett’s t-test for comparison with the control group. Differences were considered significant at P < 0.05. 3. Results and discussion 3.1. Physicochemical characterization Table 2 summarizes the main physicochemical properties of the nanoparticles used in this study. Interestingly, the mean size of empty nanoparticles appeared to be dependent on the desolvation process used to obtain the nanoparticles. In all cases, the polydispersity index (PDI) were low and below 0.2, indicating homogeneous formulations. Nanoparticles prepared by the desolvation of the polymer in acetone with a mixture of ethanol and water yielded higher mean diameters than those obtained in the presence of water (250 nm vs. 180 nm). This fact would be related to the polarity of solvents used to prepare the nanoparticles. In this particular case, the polymer (PVM/MA) is

Table 2 Physicochemical properties of nanoparticles used in this study. PDI: polydispersity index. See Table 1 for abbreviations of nanoparticles formulation. Size (nm)

SD EtOH SD H2O LF EtOH LF H2O

PDI

Zeta Potential (mV)

Empty

With lumogen

Empty

With lumogen

251 186 242 174

257 170 258 179

0.115 0.121 0.126 0.133

0.161 0.158 0.171 0.121

Empty 37 31 50 37

With lumogen 46 37 49 43

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dissolved in acetone (dielectric constant: 20.7; polarity index: 5.1) (Hansen, 2007). The addition of solvents with lower polarity than acetone (e.g. ethanol and water) induces the development of hydrophobic interactions between the polymer chains and, thus, its precipitation in the form of nanoparticles (Galindo-Rodriguez et al., 2004). Water displays a higher polarity (dielectric constant: 80.1; polarity index: 10.2) (Hansen, 2007) than ethanol (dielectric constant: 24.5; polarity index: 6.0) (Hansen, 2007). Thus, the addition of water to the solution of the polymer in acetone would induce a more abrupt desolvation of the polymer and the formation of smaller nanoparticles than with the addition of ethanol. Apart the polarity, another factor that may influence the size of nanoparticles is the viscosity of the solvents employed in their preparation (Galindo-Rodriguez et al., 2004; Honary and Zahir, 2013). In fact, ethanol displays a slightly higher viscosity than water (1.2 vs. 1.0Cp). Thus, in our case, the desolvation of the polymer with the ethanol:water mixture would provide a higher mass transfer resistance and, therefore, the diffusion of polymersolvent phase into the external aqueous phase is reduced, leading to a higher size of the resultant nanoparticles. In addition, the nanoparticles prepared in the presence of water showed a negative zeta potential slightly lower (in absolute value) than nanocarriers prepared with the mixture of ethanolwater. However, the step that appeared to influence the surface charge of nanoparticles was the drying procedure. Thus, nanoparticles dried by lyophilization presented higher negative zeta potentials than those dried by spray-drying ( 47 mV vs. 35 mV). The presence of sugars, included to facilitate the drying step and

re-dispersion (sucrose in lyophilisation, or mannitol in spraydrying), can affect both the viscosity and refractive index of the dispersant and, then, modify the zeta potential data. However, more studies are needed to clearly elucidate this aspect. Finally, it is also interesting to highlight that the labelling of nanoparticles with Lumogen red (see Section 3.3) only slightly modified the physicochemical properties of the resulting nanocarriers. 3.2. Cytotoxicity To establish an optimal protocol for the evaluation of the different formulations, it was crucial to evaluate their in vitro cellular toxicity. We investigated the viability of J744 cells after exposure to the four PVM/MA nanoparticle formulations: SD H2O, SD EtOH, LF H2O or LF EtOH (Table 1). Different concentrations of each formulation (from 25 to 400 mg/mL) for 3 or 24 h were tested and cellular toxicity was subsequently assessed with the MTT assay. Cytotoxicity, expressed as a percentage of cell viability, was calculated from the measured absorbance values normalize to the negative control (100% viability). Results are shown in Fig. 1. The data indicated that nanoparticles based on PVM/MA were not cytotoxic. Only a slight decrease in the viability of cells was observed after exposure to SD EtOH nanoparticles for 24 h; although, this effect was not dose dependent. With the other three formulations (SD H20, LF EtOH or LF H2O), no effect on the viability of J774 cells was observed after either 3 or 24 h.

A

% viavility

150

100

50

0

% viavility

B

25 10 0 20 0 400 SD EtOH

25 10 0 20 0 400 SD H2O

25 10 0 20 0 400 LF EtOH

25 10 0 20 0 400 LF H2O

25 10 0 20 0 400

25 10 0 20 0 400

25 10 0 20 0 400

25 10 0 20 0 400

SD H2O

LF EtOH

150

100

50

0 SD EtOH

LF H2O

NPs concentration (ug/mL) Fig. 1. Viability of J774 cells after exposure to PVM/MA nanoparticles for either 3 (A) or 24 h (B) determined by MTT assay. Data are expressed as mean +/ SD; n = 3 for each data point. See Table 1 for abbreviations of nanoparticles formulation.

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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3.3. Uptake kinetics of nanoparticles Slight physicochemical differences may lead to significant implications in the cellular uptake and biological processing of nanoparticles (Alexis et al., 2008; Verma and Stellacci, 2010). As a consequence, studies related with the in vitro fate of nanoparticles require uniformity of particle size and surface charge in order to establish accurate comparisons on cellular uptake. In our case, important differences were found related to size between water and ethanol:water nanoparticles. However, since all conditions in the desolvation process were identical (temperature, stirring, flow, etc.), it was still well-meaning to compare their uptake differences for our final purpose of using them in vaccination. First, it was studied the uptake of lumogen nanoparticles into J774 macrophages for 24 h at different concentrations, from 25 to 400 mg/mL. Fig. 2 shows the median of fluorescence values of the cells determined by flow cytometry after the corresponding incubation time. Nanoparticles prepared in the presence of water (LF H2O and SD H2O) were more efficiently internalized by cells than nanoparticles prepared with the mixture of ethanol and water (LF EtOH and SD EtOH). Thus, at a nanoparticle concentration of 100 mg/mL, nanoparticles produced in the presence of water displayed a capability to be taken up by macrophages at least 3-times higher than that observed for nanoparticles prepared with the hydroalcoholic solution (Fig. 3). This behaviour could be related to the differences in size, showing an inverse relationship between the size and the capability of phagocytic cells to internalize nanoparticles (Murugan et al., 2015; Nicolete et al., 2011; Loh et al., 2012). Regarding cellular uptake of nanoparticles, another important aspect would be the surface charge of these carriers. Thus, LF EtOH nanoparticles (with a zeta potential of 50 mV) displayed the lowest efficiency to be internalized by phagocytic cells (see Figs. 2 and 3). The relation between the surface charge of nanoparticles and their interaction with cells has been described by several groups (Verma and Stellacci, 2010). As a general view, the uptake of nanoparticles by cells can be viewed as a two-step process: first, a binding step on the cell membrane and second, the internalization step (Ciani et al., 2007). The attachment of nanoparticles to cell membrane seems to be most affected by the surface charge of the particles (Chen et al., 2010; Patila et al., 2007). Accordingly, apart from size and surface charge, other

Fig. 3. Uptake of nanoparticles by J744 cells. Cells were exposed to the lumogenloaded nanoparticles at a final concentration of 100 mg/mL for either 3 or 24 h, and analysed by flow cytometry. Mean values and standard deviations of three triplicates are given. Results are reported as fluorescence values. See Table 1 for abbreviations of nanoparticles formulation.

physicochemical properties must be considered to explain the differences in the cellular uptake process. Shape is one of the primary parameters that also requires attention. The vast majority of nanoparticles developed for drug delivery have a spherical shape, but other forms such as cube-shaped (so-called cubosomes), cylindrical, ellipsoids, and disks have recently been proposed as new drug nanocarriers (Agarwal et al., 2013). However, this factor cannot be considered as a discriminatory factor since the formulations studied here present round shape (Ojer et al., 2012). Rigidity seems to be a further significant factor influencing the entry pathway. It has been showed macrophages tend to present a strong preference for rigid particles because soft ones are unable to stimulate the formation and closure of phagosomes (Beningo and Wang, 2002). Consequently, further studies concerning the rigidity of our formulation could give us some light about its different behaviour. Finally, the initial uptake of nanoparticles by J774 cells was correlated with the concentration of nanoparticles (see Fig. 2). Nevertheless, after 24 h of incubation, a plateau effect was

Fluorescence intensity

200000

LF H2O 150000

100000

SD H2O 50000

SD EtOH LF EtOH

0

25

100

200

400

NPs concentration (µg/mL) Fig. 2. Cellular uptake of nanoparticles by J744 cells. Cells were exposed to the lumogen-loaded nanoparticles at a final concentration of 25, 100, 200 or 400 mg/mL for 24 h, then fixed and analysed by flow cytometry. Data are expressed as mean +/ SD; n = 3 for each data point. Results are reported as fluorescence values. Background fluorescence was corrected by including controls to measure the fluorescence from cultures in the absence of nanoparticles. See Table 1 for abbreviations of nanoparticles formulation. See Table 1 for abbreviations of nanoparticles formulation.

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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observed. That could be due to the limited saturation level. In other words, the plateau effect of the cellular uptake efficiency suggests this uptake is saturable. These findings are compatible with the observations from other groups being related with the active mechanistic uptake process that is discussed below (Derakhshandeh et al., 2011). 3.4. Respiratory burst The “respiratory burst” involving production of superoxide anions is one of the marked responses of macrophage stimulation. Therefore, to determine the production and accumulation of intracellular reactive oxygen species (ROS) induced by the different nanoparticle formulations, we chose to use the ROS-sensitive dye dihydrorhodamine (DHR-123), which has been used previously as a general indicator of cellular ROS levels. Multiple ROS directly oxidize DHR-123 to the highly stable, fluorescent derivative rhodamine-123 in such a way that an increase in the fluorescent signal reflects ROS production (Sakurada et al., 1992). Fig. 4 summarizes the relative activation of oxidative burst in the cells, by showing the fluorescence value of rhodamine. The more DHR was oxidized, the higher was the signal. As it was expected, control cells treated with PMA showed the highest median value of fluorescence value. Cells treated with nanoparticles (and pre-treated with DHR) demonstrated a similar fluorescence value as the negative control (p < 0.05), suggesting that nanoparticles did not induce ROS under these experimental conditions. 3.5. Pharmacological inhibitors To further understand the specific mechanisms involved in the cellular internalization of PVM/MA nanoparticles, four pharmacological inhibitors were used to interfere with various uptake pathways. Inhibitors were chosen based on their selectivity and applications (Dutta and Donaldson, 2012; Vercauteren et al., 2010). Considering that some internalization pathways share some mechanisms and machinery with other pathways, inhibitors used in this study were used alone or in combination, in order to be more accurate for a particular mechanism. Besides, it should be noted that inhibitory effects of these agents are cell-typedependent (Vercauteren et al., 2010) and, accordingly, conclusions from this study are limited to J774 macrophages. Inhibitor

concentrations were optimized to achieve a minimum of 90% cell viability over 8 h for J744 cells. For all inhibition studies, particles were administered for 3 h. The prolongation of the exposure to these inhibitors usually induces the death of cells (Agarwal et al., 2013). Fig. 5 shows the effects of different inhibitors on the uptake of nanoparticles by J744 cells. Cytochalasin D, a blocking agent of the actin polymerization that inhibits membrane ruffling and macopinocytosis, decreased the cellular uptake of all types of nanoparticles in a similar extent. Thus, the incubation of cells with 5 mM cytochalasin decreased by approximately 50% the uptake of PVM/MA nanoparticles by J744 cells. Chlorpromazine, a cationic drug that results in loss of clathrin and AP2 adaptor complex protein from the cell surface, blocks specifically the clathrin-mediated pathway (Wang et al., 1993). When chlorpromazine was used, nanoparticles obtained in water (SD H2O and LF H2O) displayed a significantly lower uptake by J744 cells than those prepared with a mixture of ethanol and water. These results confirm the role of actin in the engulfment process, suggesting that clathrin-mediated pathway is used by PVM/MA nanoparticles to enter into the phagocytic cells, in particular those with a lower diameter (SD H2O and LF H2O). The role of actin in the engulfment process was confirmed by using the combination of chlorpromazine and cytochalasin, leading to an important inhibition of the phagocytic process (Fig. 5). To confirm these results, the effect of LY294002 and wortmannin, specific inhibitors for pathways depending on actin (e.g., macropinocytosis and phagocytosis) (Vlahos et al., 1994), were evaluated. When LY294002 was used alone, discrete decreases of nanoparticle uptake were found, as compared with control (Fig. 5). However, when this inhibitor was used in combination with wortmannin, the amount of nanoparticles with a lower mean diameter (SD H2O and LF H2O) displayed a significant decrease in their capture and internalization by J744 cells. Taken all together, these results indicate that PVM/MA nanoparticles are internalized by active endocytosis mediated by actin. For PVM/MA nanoparticles prepared by the addition of water which present sizes around 180 nm, the inhibition studies indicated that J774 cells use multiple uptake pathways such as macropinocytosis, membrane ruffling and clathrin-mediated pathway. In the case of nanoparticles prepared by the addition of ethanol:water, cells internalized these nanoparticles specially by clathrin-mediated pathway and in less proportion by macropinocytosis. Regarding to the nanoparticles evaluated in this study, it seems that surface charge is not of particular relevance since no differences between formulations, using the same desolvating agent, were observed. Thus, the critical factor that influences the entrance of these PVM/MA nanoparticles would be the size. 4. Remarks on vaccination

Fig. 4. Respiratory burst in J744 cells. J774 were preincubated with DHR and, thereafter, non-activated (basal conditions, &), stimulated by phorbol myristate acetate (PMA (&) as a positive control), or incubated with the nanoparticle formulations at a final concentration of 100 mg/mL. See Table 1 for abbreviations of nanoparticles formulation. Results expressed as fluorescence intensity (mean +/ SD; n = 3). Student’s t-test was performed to discriminate among effects of different cell treatments (*p < 0.05 vs. basal conditions).

Adaptive immunity requires activation of T lymphocytes by APC, which presents antigen bound by major histocompatibility complex (MHC) molecules which depends on the ability of APC to display peptide-MHC complexes at their surface. The study of the interaction between nanoparticles and antigen-presenting cells (APCs) is of particular interest in the field of vaccinology. It is assumed that antigens which are displayed intracellularly are presented by Class I MHC molecules and activate cytotoxic CD8+ T cells. In contrast, extracellular antigens are presented by Class II MHC molecules to activate CD4+ T helper cells. A further mechanism termed cross-presentation permits some forms of extracellular antigen to also stimulate CD8+ T cells via the MHC I pathway. Particularly, cross-presentation is

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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Fig. 5. Effect of transport inhibitors on the internalization of PVM/MA nanoparticles into J744 cells. Cells were pretreated with each one of the inhibitors (cytochalasin D, LY940002, wortmannin or chlorpromazine) for 30 min, followed by 3 h of exposure to the nanoparticles (100 mg/mL), in the presence of the same inhibitor, then fixed and analysed by flow cytometry. Results, reported as the percentage of uptake relative to untreated cells exposed to nanoparticles with no inhibitors, are expressed as mean +/ SD, n = 3. See Table 1 for abbreviations of nanoparticles formulation.

linked to cytotoxic T cell (CTL) responses required in protection against intracellular pathogens and tumour cells (Neefjes and Sadaka, 2012). Potent CTL responses have been reported to be limited to live attenuated viral or bacterial vaccines (Esser et al., 2003; Kim et al., 2010). However, the use of such vaccines is offset by the risk of reinitiating virulence. The potential of APCs to crosspresent antigen has initiated many research questions aimed at finding strategies to enhance cross-presentation in DCs. Thus, the fate for a particular antigen relies on the ability of cells to deliver the antigens to the correct processing compartments. Several factors are involved in this process such as the type of antigen, the presence of cell stimulatory factors, which can be altered adjuvants, and the timing and phase of the immune response. Since there is no a particular feature that is necessary and sufficient, the addition of several “necessary factors” can lead to the expected ending. First, it is generally believed that the mechanism by which antigens are captured by cells is important for cross-presentation. Moreover, the form of the antigen, its solubility, and whether it is part of an immune complex or still associated with a pathogen all determine the route of entry. In general terms, particulate antigens are captured by phagocytosis and soluble antigens by pinocytosis. Several works indicate that antigens internalized by phagocytosis have long been known to be much more efficiently cross-presented than those internalized by fluid-phase endocytosis (Tran and Shen, 2009). Thus, particulate antigens could be subsequent expressed by Class I MHC molecules in a higher rate than soluble antigens. Accordingly, the results presented here stating that PVM/MA nanoparticles are internalized by clathrin-mediated pathways, rather than caveoladependent endocytosis, would contribute to the antigen cross presentation pathway. In fact, these results support our previous findings that PVM/MA nanoparticles induce potent cytotoxic immune response against the encapsulated antigens (Tamayo et al., 2010). Our hypothesis is that antigens containing PVM/MA nanoparticles enter the early endosome via clathrin-mediated endocytosis and differentially traffic into the late endosome. From here, the antigen escape from the endosome to the cytosol to prime CD8+ T cells through cross-presentation. Thus, a lot of work is being focused on the study of different polymeric nanoparticles in order to evaluate the different mechanism that can lead to this particular immune response (Elsabahy and wooley, 2012; Kamaly et al., 2012). Second, in addition to inducing cytokine secretion and the upregulation of costimulatory molecules, innate receptor signaling

may also contribute to the regulation of cross-presentation. For example, a large set of TLR ligands are known to act as adjuvants and stimulate cross-presentation (Pooley et al., 2001; Jongbloed et al., 2010; Poulin et al., 2010; Kool et al., 2011; Mouries et al., 2008), illustrating that the antigen-presenting capability of different DC subsets is dependent on the stimulatory agent used. For instance, it has been established a mechanism by which TLR4 or TLR2 might regulate cross-presentation of exogenous antigens to induce CD8+ T cell priming (Nair-Gupta and Blander, 2013; Kuan et al., 2014). Remarkably, previous work of our group has described how PVM/MA nanoparticles are capable of activating TLR-2 and TLR-4 (Tamayo et al., 2010). Thus, this could be another event that could make PVM/MA polymeric nanoparticles to trigger cross-presentation of a particular antigen. Further studies to confirm such hypothesis are important since information on the mechanisms involved will help us to develop different polymeric nanoparticles that can lead to this particular immune response (Elsabahy and Wooley, 2012; Kamaly et al., 2012). 5. Conclusions In the present study, PVM/MA nanoparticles were evaluated. First, we studied the effect of the desolvation process, comparing the employment of water or a mixture of ethanol:water in order to form nanoparticles. Second, we tested the drying process of the nanoparticles, comparing lyohilization and spray-drying. Third, we evaluated the interaction of PVM/MA nanoparticles with the cell line of macrophages J774. Taken all the results together, we can conclude that the desolvation process has an important effect on the size of resultant nanoparticles. Thus, nanoparticle prepared by the addition of water present a size smaller than those formed by the addition of water:ethanol. Besides, it seems the drying process has a slightly impact on the surface charge. When using macrophages 774 cells, it was observed that PVM/ MA nanoparticles has no toxic effect. Besides, looking for the entrance mechanism of these nanoparticles into the cells, results indicated that PVM/MA nanoparticles prepared by the addition of water which present sizes around 180 nm, use multiple uptake pathways such as macropinocytosis, membrane ruffling and clathrin-mediated pathway. In the case of nanoparticles prepared by the addition of ethanol:water, cells internalized these nanoparticles specially by clathrin-mediated pathway and in less proportion by macropinocytosis.

Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030

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Please cite this article in press as: Gamazo, C., et al., Interactions of poly (anhydride) nanoparticles with macrophages in light of their vaccine adjuvant properties. Int J Pharmaceut (2015), http://dx.doi.org/10.1016/j.ijpharm.2015.10.030